Team:UT-Tokyo/LabNote

From 2011.igem.org

(Difference between revisions)
Line 213: Line 213:
*Planting Negative control
*Planting Negative control
*Making frozen stock
*Making frozen stock
-
*Transformation(BBa_E0240,B0032,B0015,B0014,E0040,J31000,J44000)
+
*Transformation(BBa_E0240, <nowiki>#</nowiki>3, B0015, <nowiki>#</nowiki>5, <nowiki>#</nowiki>10, ,J31000, ,J44000)
=='11/06/14(Tue)==
=='11/06/14(Tue)==
Line 223: Line 223:
=='11/06/17(Fri)==
=='11/06/17(Fri)==
-
*Picking up colony(B0032,B0015,B0040,E0240,J31000,J44000)
+
*Picking up colony(<nowiki>#</nowiki>3, B0015, B0040,<nowiki>#</nowiki>9, J31000, J44000)
-
*Miniprep(B0014)
+
*Miniprep(<nowiki>#</nowiki>5)
-
*Dissolution(J23119,J23118,K16500,J22106)
+
*Dissolution(<nowiki>#</nowiki>1, <nowiki>#</nowiki>2, K16500, <nowiki>#</nowiki>24)
=='11/06/21(Tue)==
=='11/06/21(Tue)==
-
*Making frozen stock(B0032,B0015,B0040,E0240,J31000,J44000)
+
*Making frozen stock(<nowiki>#</nowiki>3, B0015, B0040, <nowiki>#</nowiki>9, J31000, J44000)
-
*Passafe culture(E0240,J31000)
+
*Passafe culture(<nowiki>#</nowiki>9, J31000)
*Nanodrop again
*Nanodrop again
=='11/06/22(Wed)==
=='11/06/22(Wed)==
-
*Miniprep(E0240,J31000)
+
*Miniprep(<nowiki>#</nowiki>9, J31000)
-
*Making Competent cell
+
*Making competent cell
=='11/06/24(Fri)==
=='11/06/24(Fri)==
-
*Digest (product of Miniprep 06/09)(EScut)
+
*Digest (product of Miniprep 06/09 EScut)
*Making agarose gel
*Making agarose gel
*Electrophoresis
*Electrophoresis
=='11/06/28(Tue)==
=='11/06/28(Tue)==
-
*Digest(product of Miniprep 06/09)(EPcut)
+
*Digest(product of Miniprep 06/09 EPcut)
*Electrophoresis
*Electrophoresis
-
*Defrost and transformation(BBa_E0030,E0020,I712019,I712052,J52008）
+
*Defrost and transformation(BBa_E0030, E0020, <nowiki>#</nowiki>14, I712052,<nowiki>#</nowiki>17）
*Making 1×TAE, agarose gel, LB medium, LB plate(amp),
*Making 1×TAE, agarose gel, LB medium, LB plate(amp),
Line 253: Line 253:
=='11/07/01(Fri)==
=='11/07/01(Fri)==
*Digest(EPcut)
*Digest(EPcut)
-
*Miniprep(BBa_E0030,E0020,I712019,I712052,J52008)
+
*Miniprep(BBa_E0030, E0020, <nowiki>#</nowiki>14, I712052, <nowiki>#</nowiki>17)
=='11/07/05(Tue)==
=='11/07/05(Tue)==
*Electrophoresis(product of digest 07/01)
*Electrophoresis(product of digest 07/01)
-
*Digest(E0240,I712019,J52008,B0032,B0014)
+
*Digest(<nowiki>#</nowiki>9, <nowiki>#</nowiki>14, <nowiki>#</nowiki>17, <nowiki>#</nowiki>3, <nowiki>#</nowiki>5)
*Making antibiotic stock(1000×)(Km,Cm)
*Making antibiotic stock(1000×)(Km,Cm)
=='11/07/06(Wed)==
=='11/07/06(Wed)==
*Gel  extraction (pre)(BBa_I712052)
*Gel  extraction (pre)(BBa_I712052)
-
*Picking up colony(Ba_J23119,J23118,J22106,E0040?)
+
*Picking up colony(Ba_J23119, <nowiki>#</nowiki>2, <nowiki>#</nowiki>24, <nowiki>#</nowiki>10)
*Making agarose gel
*Making agarose gel
=='11/07/07(Thu)==
=='11/07/07(Thu)==
-
*Digest（BBa_E0240,I712019,J52008,B0032,B0014）
+
*Digest（BBa_E0240, <nowiki>#</nowiki>14, <nowiki>#</nowiki>17, <nowiki>#</nowiki>3, <nowiki>#</nowiki>5）
-
*Defrost（BBa_E1010,K325101,K145001,I712074,R0011,C0012）
+
*Defrost（BBa_E1010, K325101, <nowiki>#</nowiki>20, <nowiki>#</nowiki>21, <nowiki>#</nowiki>22, <nowiki>#</nowiki>23）
-
*Transformation（BBa_K145001,I712074,R0011,C0012）
+
*Transformation（BBa_K145001, <nowiki>#</nowiki>20, <nowiki>#</nowiki>21, <nowiki>#</nowiki>22）
-
*Making LB plate（Cm×10,Km×9）
+
*Making LB plate（Cm,Km）
=='11/07/08(Fri)==
=='11/07/08(Fri)==
-
*Miniprep(BBa_J23119,J23118,J22106,E0040)
+
*Miniprep(BBa_J23119, <nowiki>#</nowiki>2, <nowiki>#</nowiki>10, <nowiki>#</nowiki>24)
=='11/07/11(Mon)==
=='11/07/11(Mon)==
-
*Gel extraction(E0240,I712019,J52008)
+
*Gel extraction(<nowiki>#</nowiki>9 <nowiki>#</nowiki>14, <nowiki>#</nowiki>17)
-
*Transformation(J23119,R0011,C0012,E1010,K325101,K145001,I712074)
+
*Transformation(<nowiki>#</nowiki>1, <nowiki>#</nowiki>11, <nowiki>#</nowiki>20, <nowiki>#</nowiki>21, K325101, <nowiki>#</nowiki>22, <nowiki>#</nowiki>23)
=='11/07/12(Tue)==
=='11/07/12(Tue)==
Line 282: Line 282:
=='11/07/13(Wed)==
=='11/07/13(Wed)==
-
*Frozen stock(J23119(18A),R0011(6G),C0012(2O),I712079(6N),K145001(2F))
+
*Frozen stock(<nowiki>#</nowiki>1(18A), <nowiki>#</nowiki>20(6G), <nowiki>#</nowiki>21(2O), <nowiki>#</nowiki>14(6N), <nowiki>#</nowiki>23(2F))
-
*Picking up colony(E1010)
+
*Picking up colony(<nowiki>#</nowiki>11)
-
*Gel extraction(B0032,B0014)
+
*Gel extraction(B<nowiki>#</nowiki>3, <nowiki>#</nowiki>5)
-
*Digest (J23118 SPcut)
+
*Digest (<nowiki>#</nowiki>2 SPcut)
=='11/07/14(Thu)==
=='11/07/14(Thu)==
-
*Miniprep(E1010,J23119,K145001,I712074,R0011,C0012)
+
*Miniprep(<nowiki>#</nowiki>1, <nowiki>#</nowiki>11, <nowiki>#</nowiki>20, <nowiki>#</nowiki>21, <nowiki>#</nowiki>22, <nowiki>#</nowiki>23)
-
*Electrophoresis(J23118)
+
*Electrophoresis(<nowiki>#</nowiki>2)
-
*Gel extraction(J23118,B0032,B0014)
+
*Gel extraction(<nowiki>#</nowiki>2, <nowiki>#</nowiki>3, <nowiki>#</nowiki>5)
-
*Passafe(E1010)
+
*Passafe(<nowiki>#</nowiki>11)
*Making LB+amp
*Making LB+amp
=='11/07/15(Fri)==
=='11/07/15(Fri)==
-
*Ligation(J23118-E0240,J23118-B0032,I712019-B0014,J52008-B0014)
+
*Ligation(<nowiki>#</nowiki>2-<nowiki>#</nowiki>9, <nowiki>#</nowiki>2-<nowiki>#</nowiki>3, <nowiki>#</nowiki>14-<nowiki>#</nowiki>5, <nowiki>#</nowiki>17-<nowiki>#</nowiki>5)
-
*Transformation(J23118-E0240,J23118-B0032,I712019-B0014,J52008-B0014)
+
*Transformation(<nowiki>#</nowiki>2-<nowiki>#</nowiki>9, <nowiki>#</nowiki>2-<nowiki>#</nowiki>3, <nowiki>#</nowiki>14-<nowiki>#</nowiki>5, <nowiki>#</nowiki>17-<nowiki>#</nowiki>5)
*Dispensing Ligation Buffer
*Dispensing Ligation Buffer
-
*Frozen stock(E1010)
+
*Frozen stock(<nowiki>#</nowiki>11)
=='11/07/19(Tue)==
=='11/07/19(Tue)==
Line 309: Line 309:
*Making Master plate
*Making Master plate
*Testing product of Miniprep
*Testing product of Miniprep
-
*Transformation (<nowiki>#</nowiki>2,<nowiki>#</nowiki>27,<nowiki>#</nowiki>28)
+
*Transformation (<nowiki>#</nowiki>2, <nowiki>#</nowiki>27, <nowiki>#</nowiki>28)
=='11/07/21(Thu)==
=='11/07/21(Thu)==
-
*Miniprep(<nowiki>#</nowiki>2-3,<nowiki>#</nowiki>2-9,<nowiki>#</nowiki>14-5,<nowiki>#</nowiki>17-5,<nowiki>#</nowiki>11,<nowiki>#</nowiki>20)
+
*Miniprep(<nowiki>#</nowiki>2-3, <nowiki>#</nowiki>2-9, <nowiki>#</nowiki>14-5, <nowiki>#</nowiki>17-5, <nowiki>#</nowiki>11, <nowiki>#</nowiki>20)
*Testing product
*Testing product
-
*Digest(<nowiki>#</nowiki>20,<nowiki>#</nowiki>2-3 SPcut　<nowiki>#</nowiki>10,<nowiki>#</nowiki>11,<nowiki>#</nowiki>14-5,<nowiki>#</nowiki>17-5 XPcut）
+
*Digest
 +
**SPcut : <nowiki>#</nowiki>20, <nowiki>#</nowiki>2-3
 +
**XPcut : <nowiki>#</nowiki>10, <nowiki>#</nowiki>11, <nowiki>#</nowiki>14-5, <nowiki>#</nowiki>17-5
*Making TB
*Making TB
=='11/07/22(Fri)==
=='11/07/22(Fri)==
-
*Frozen stock(<nowiki>#</nowiki>2,<nowiki>#</nowiki>2-3,<nowiki>#</nowiki>2-9,<nowiki>#</nowiki>14-5,<nowiki>#</nowiki>17-5）
+
*Frozen stock(<nowiki>#</nowiki>2, <nowiki>#</nowiki>2-3, <nowiki>#</nowiki>2-9, <nowiki>#</nowiki>14-5, <nowiki>#</nowiki>17-5）
-
*Electrophoresis(<nowiki>#</nowiki>20,<nowiki>#</nowiki>2-3 SPcut　<nowiki>#</nowiki>10,<nowiki>#</nowiki>11,<nowiki>#</nowiki>14-5,<nowiki>#</nowiki>17-5 XPcut）
+
*Electrophoresis(<nowiki>#</nowiki>20_SP, <nowiki>#</nowiki>2-3_SP, <nowiki>#</nowiki>10_XP, <nowiki>#</nowiki>11_XP, <nowiki>#</nowiki>14-5_XP, <nowiki>#</nowiki>17-5_XP）
-
*Digest(<nowiki>#</nowiki>20 SPcut, <nowiki>#</nowiki>10,<nowiki>#</nowiki>11,<nowiki>#</nowiki>14-5,<nowiki>#</nowiki>17-5 XPcut)
+
*Digest
 +
**SPcut : <nowiki>#</nowiki>20
 +
**XPcut : <nowiki>#</nowiki>10, <nowiki>#</nowiki>11, <nowiki>#</nowiki>14-5, <nowiki>#</nowiki>17-5
*Transformation(<nowiki>#</nowiki>27, <nowiki>#</nowiki>28, product of Miniprep 07/20)
*Transformation(<nowiki>#</nowiki>27, <nowiki>#</nowiki>28, product of Miniprep 07/20)
=='11/07/25(Mon)==
=='11/07/25(Mon)==
-
*Colony check(<nowiki>#</nowiki>2,<nowiki>#</nowiki>2-9)
+
*Colony check(<nowiki>#</nowiki>2, <nowiki>#</nowiki>2-9)
-
*Gel Extraction(<nowiki>#</nowiki>20 SPcut <nowiki>#</nowiki>10,<nowiki>#</nowiki>11,<nowiki>#</nowiki>14-5,<nowiki>#</nowiki>17-5 XPcut)
+
*Gel Extraction(<nowiki>#</nowiki>20_SP, <nowiki>#</nowiki>10_XP, <nowiki>#</nowiki>11_XP, <nowiki>#</nowiki>14-5_XP, <nowiki>#</nowiki>17-5_XP)
-
*Ligation and Transformation()
+
*Ligation and Transformation(<nowiki>#</nowiki>20-<nowiki>#</nowiki>9, <nowiki>#</nowiki>3-<nowiki>#</nowiki>11, <nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5, <nowiki>#</nowiki>3-<nowiki>#</nowiki>17-5)
*Defrost Primer(200×, 10×)
*Defrost Primer(200×, 10×)
*Dispensing PCR Mix
*Dispensing PCR Mix
=='11/07/26(Tue)==
=='11/07/26(Tue)==
-
*Picking up colony and making master plate(<nowiki>#</nowiki>20-9,<nowiki>#</nowiki>3-11,<nowiki>#</nowiki>3-14-5,<nowiki>#</nowiki>3-17-5)
+
*Picking up colony and making master plate(<nowiki>#</nowiki>20-9, <nowiki>#</nowiki>3-11, <nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>3-17-5)
-
*Colony PCR(<nowiki>#</nowiki>20-9,<nowiki>#</nowiki>3-11,<nowiki>#</nowiki>3-14-5,<nowiki>#</nowiki>3-17-5)
+
*Colony PCR(<nowiki>#</nowiki>20-9, <nowiki>#</nowiki>3-11, <nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>3-17-5)
*Digest(<nowiki>#</nowiki>14-5 XPcut,Ecut, <nowiki>#</nowiki>22 SPcut, <nowiki>#</nowiki>23 XPcut)
*Digest(<nowiki>#</nowiki>14-5 XPcut,Ecut, <nowiki>#</nowiki>22 SPcut, <nowiki>#</nowiki>23 XPcut)
*Ligation(<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5(Re), <nowiki>#</nowiki>3(Negative control))
*Ligation(<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5(Re), <nowiki>#</nowiki>3(Negative control))
-
*Transformation(<nowiki>#</nowiki>15,<nowiki>#</nowiki>27,<nowiki>#</nowiki>28,<nowiki>#</nowiki>30,<nowiki>#</nowiki>3-14-5(Re), <nowiki>#</nowiki>3(Negative control))
+
*Transformation(<nowiki>#</nowiki>15, <nowiki>#</nowiki>27, <nowiki>#</nowiki>28, <nowiki>#</nowiki>30, <nowiki>#</nowiki>3-14-5(Re), <nowiki>#</nowiki>3(Negative control))
-
*Making reagent for TB(KOH,CaCl2,KCl,MnCl2)
+
*Making reagent for TB(KOH, CaCl2, KCl, MnCl2)
=='11/07/27(Wed)==
=='11/07/27(Wed)==
-
*Miniprep(<nowiki>#</nowiki>1,<nowiki>#</nowiki>2,<nowiki>#</nowiki>3,<nowiki>#</nowiki>10,<nowiki>#</nowiki>22,<nowiki>#</nowiki>24,<nowiki>#</nowiki>20-9,<nowiki>#</nowiki>3-11,<nowiki>#</nowiki>3-17-5)
+
*Miniprep(<nowiki>#</nowiki>1, <nowiki>#</nowiki>2, <nowiki>#</nowiki>3, <nowiki>#</nowiki>10, <nowiki>#</nowiki>22, <nowiki>#</nowiki>24, <nowiki>#</nowiki>20-9, <nowiki>#</nowiki>3-11, <nowiki>#</nowiki>3-17-5)
*Electrophoresis
*Electrophoresis
=='11/07/28(Thu)==
=='11/07/28(Thu)==
-
*Gel extraction(<nowiki>#</nowiki>14-5 XPcut, <nowiki>#</nowiki>22 SPcut, <nowiki>#</nowiki>23 XPcut)
+
*Gel extraction(<nowiki>#</nowiki>14-5_XP, <nowiki>#</nowiki>22_SP, <nowiki>#</nowiki>23_XP)
-
*Digest(<nowiki>#</nowiki>1,<nowiki>#</nowiki>2,<nowiki>#</nowiki>3,<nowiki>#</nowiki>24 SPcut, <nowiki>#</nowiki>3-11 EScut, <nowiki>#</nowiki>3-17-5 XPcut)
+
*Digest
 +
**SPcut : <nowiki>#</nowiki>1,<nowiki>#</nowiki>2, <nowiki>#</nowiki>3, <nowiki>#</nowiki>24
 +
**EScut : <nowiki>#</nowiki>3-11
 +
**XPcut : <nowiki>#</nowiki>3-17-5
*Ligation(<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5)
*Ligation(<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5)
*Making TB bugger, LB plate, 0.3% LB plate
*Making TB bugger, LB plate, 0.3% LB plate
=='11/07/29(Fri)==
=='11/07/29(Fri)==
-
*Cloning(lexA,cheZ)
+
*Cloning(lexA, cheZ)
*Colony PCR(<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5)
*Colony PCR(<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5)
-
*Gel extraction and Agarase処理　(<nowiki>#</nowiki>1,<nowiki>#</nowiki>2,<nowiki>#</nowiki>3,<nowiki>#</nowiki>24 SPcut, <nowiki>#</nowiki>3-11 EScut, <nowiki>#</nowiki>3-17-5 XPcut, lexA,cheZ PCR product）
+
*Gel extraction(<nowiki>#</nowiki>1_SP, <nowiki>#</nowiki>2_SP, <nowiki>#</nowiki>3_SP, <nowiki>#</nowiki>24_SP, <nowiki>#</nowiki>3-11_ES, <nowiki>#</nowiki>3-17-5_XP, lexA, cheZ PCR product）
*Miniprep(<nowiki>#</nowiki>30)
*Miniprep(<nowiki>#</nowiki>30)
*Digest(lexA, cheZ PCR product XP cut)
*Digest(lexA, cheZ PCR product XP cut)
=='11/08/01(Mon)==
=='11/08/01(Mon)==
-
*Nanodrop(<nowiki>#</nowiki>3-11(1),<nowiki>#</nowiki>1,<nowiki>#</nowiki>3,<nowiki>#</nowiki>24,<nowiki>#</nowiki>2,<nowiki>#</nowiki>3-17-5(4)M<nowiki>#</nowiki>30)
+
*Nanodrop(<nowiki>#</nowiki>3-11(1), <nowiki>#</nowiki>1, <nowiki>#</nowiki>3, <nowiki>#</nowiki>24, <nowiki>#</nowiki>2, <nowiki>#</nowiki>3-17-5(4)M<nowiki>#</nowiki>30)
-
*Digest(<nowiki>#</nowiki>3 SPcut,<nowiki>#</nowiki>5 EXcut,<nowiki>#</nowiki>9 XPcut,<nowiki>#</nowiki>30 XP cut)
+
*Digest(<nowiki>#</nowiki>3 SPcut, <nowiki>#</nowiki>5 EXcut, <nowiki>#</nowiki>9 XPcut, <nowiki>#</nowiki>30 XP cut)
-
*Cloning(lexA,cheZ)
+
*Cloning(lexA, cheZ)
-
*Gel extraction(<nowiki>#</nowiki>3,<nowiki>#</nowiki>9,<nowiki>#</nowiki>30,pSB1A2)
+
*Gel extraction(<nowiki>#</nowiki>3, <nowiki>#</nowiki>9, <nowiki>#</nowiki>30, pSB1A2)
-
*Ligation(<nowiki>#</nowiki>2-<nowiki>#</nowiki>3-17-5,<nowiki>#</nowiki>3-11-<nowiki>#</nowiki>5,<nowiki>#</nowiki>14-<nowiki>#</nowiki>5,<nowiki>#</nowiki>3-<nowiki>#</nowiki>23,<nowiki>#</nowiki>22-<nowiki>#</nowiki>9,<nowiki>#</nowiki>3-<nowiki>#</nowiki>30)
+
*Ligation(<nowiki>#</nowiki>2-<nowiki>#</nowiki>3-17-5, <nowiki>#</nowiki>3-11-<nowiki>#</nowiki>5, <nowiki>#</nowiki>14-<nowiki>#</nowiki>5, <nowiki>#</nowiki>3-<nowiki>#</nowiki>23, <nowiki>#</nowiki>22-<nowiki>#</nowiki>9, <nowiki>#</nowiki>3-<nowiki>#</nowiki>30)
-
*Transformation(<nowiki>#</nowiki>2-<nowiki>#</nowiki>3-17-5,<nowiki>#</nowiki>3-11-<nowiki>#</nowiki>5,<nowiki>#</nowiki>14-<nowiki>#</nowiki>5,<nowiki>#</nowiki>3-<nowiki>#</nowiki>23,<nowiki>#</nowiki>22-<nowiki>#</nowiki>9,<nowiki>#</nowiki>3-<nowiki>#</nowiki>30,<nowiki>#</nowiki>27(IPTG回復培養),<nowiki>#</nowiki>28)
+
*Transformation(<nowiki>#</nowiki>2-<nowiki>#</nowiki>3-17-5, <nowiki>#</nowiki>3-11-<nowiki>#</nowiki>5, <nowiki>#</nowiki>14-<nowiki>#</nowiki>5, <nowiki>#</nowiki>3-<nowiki>#</nowiki>23, <nowiki>#</nowiki>22-<nowiki>#</nowiki>9, <nowiki>#</nowiki>3-<nowiki>#</nowiki>30, <nowiki>#</nowiki>27(IPTG), <nowiki>#</nowiki>28)
=='11/08/02(Tue)==
=='11/08/02(Tue)==
-
*Colony PCR(<nowiki>#</nowiki>2-<nowiki>#</nowiki>3-17-5,<nowiki>#</nowiki>3-11-<nowiki>#</nowiki>5,<nowiki>#</nowiki>14-<nowiki>#</nowiki>5,<nowiki>#</nowiki>3-<nowiki>#</nowiki>23,<nowiki>#</nowiki>22-<nowiki>#</nowiki>9)
+
*Colony PCR(<nowiki>#</nowiki>2-<nowiki>#</nowiki>3-17-5, <nowiki>#</nowiki>3-11-<nowiki>#</nowiki>5, <nowiki>#</nowiki>14-<nowiki>#</nowiki>5, <nowiki>#</nowiki>3-<nowiki>#</nowiki>23, <nowiki>#</nowiki>22-<nowiki>#</nowiki>9)
-
*Cloning(PCR)(<nowiki>#</nowiki>27,<nowiki>#</nowiki>28)
+
*Cloning(PCR)(<nowiki>#</nowiki>27, <nowiki>#</nowiki>28)
*Gel extraction
*Gel extraction
-
*PCR_2nd(lexA,cheZ,<nowiki>#</nowiki>27,<nowiki>#</nowiki>28)
+
*PCR_2nd(lexA, cheZ, <nowiki>#</nowiki>27, <nowiki>#</nowiki>28)
*Digest (<nowiki>#</nowiki>30 again)
*Digest (<nowiki>#</nowiki>30 again)
-
*Making Competent cell(cheZ-, Wild Type)
+
*Making competent cell(cheZ-, Wild Type)
*Miniprep
*Miniprep
=='11/08/04(Thu)==
=='11/08/04(Thu)==
-
*Electrophoresis(product of ligation <nowiki>#</nowiki>2-3-17-5(2),<nowiki>#</nowiki>3-11-5(1,2),<nowiki>#</nowiki>14-5(2),<nowiki>#</nowiki>22-9(1,2,3))
+
*Electrophoresis(product of ligation <nowiki>#</nowiki>2-3-17-5(2), <nowiki>#</nowiki>3-11-5(1,2), <nowiki>#</nowiki>14-5(2), <nowiki>#</nowiki>22-9(1,2,3))
-
*Frozen stock(<nowiki>#</nowiki>2-3-17-5(2),<nowiki>#</nowiki>3-11-5(1,2),<nowiki>#</nowiki>14-5(2),<nowiki>#</nowiki>22-9(1,2))
+
*Frozen stock(<nowiki>#</nowiki>2-3-17-5(2), <nowiki>#</nowiki>3-11-5(1,2), <nowiki>#</nowiki>14-5(2), <nowiki>#</nowiki>22-9(1,2))
*Making Gel
*Making Gel
*Digest(<nowiki>#</nowiki>23 XPcut)
*Digest(<nowiki>#</nowiki>23 XPcut)
-
*IPTG induce(<nowiki>#</nowiki>20-9)
+
*IPTG induct(<nowiki>#</nowiki>20-9)
=='11/08/05(Fri)==
=='11/08/05(Fri)==
Line 386: Line 393:
*Defrost Frozen stock, Miniprep, Nanodrop and Electrophoresis(<nowiki>#</nowiki>5, <nowiki>#</nowiki>20, <nowiki>#</nowiki>23, <nowiki>#</nowiki>30)
*Defrost Frozen stock, Miniprep, Nanodrop and Electrophoresis(<nowiki>#</nowiki>5, <nowiki>#</nowiki>20, <nowiki>#</nowiki>23, <nowiki>#</nowiki>30)
*Making Gel
*Making Gel
-
*Digest(<nowiki>#</nowiki>27, <nowiki>#</nowiki>28, cheZ, lexA, <nowiki>#</nowiki>3-11-5(1), <nowiki>#</nowiki>23,<nowiki>#</nowiki>30 XPcut,<nowiki>#</nowiki>6 EScut,<nowiki>#</nowiki>7 EXcut,<nowiki>#</nowiki>7,<nowiki>#</nowiki>20 SPcut)
+
*Digest
-
*Defrost Frozen stock(<nowiki>#</nowiki>5,<nowiki>#</nowiki>14-5(2),<nowiki>#</nowiki>3-11-5(1))
+
**XPcut : <nowiki>#</nowiki>27, <nowiki>#</nowiki>28, cheZ, lexA, <nowiki>#</nowiki>3-11-5(1), <nowiki>#</nowiki>23,<nowiki>#</nowiki>30
 +
**EScut : <nowiki>#</nowiki>6
 +
**EXcut : <nowiki>#</nowiki>7
 +
**SPcut : <nowiki>#</nowiki>7,<nowiki>#</nowiki>20
 +
*Defrost Frozen stock(<nowiki>#</nowiki>5, <nowiki>#</nowiki>14-5(2), <nowiki>#</nowiki>3-11-5(1))
=='11/08/09(Tue)==
=='11/08/09(Tue)==
*Miniprep, Electrophoresis and Digest(<nowiki>#</nowiki>5, <nowiki>#</nowiki>14-5(2), <nowiki>#</nowiki>3-11-5(1))
*Miniprep, Electrophoresis and Digest(<nowiki>#</nowiki>5, <nowiki>#</nowiki>14-5(2), <nowiki>#</nowiki>3-11-5(1))
-
*Gel extraction and Nanodrop(<nowiki>#</nowiki>6,<nowiki>#</nowiki>27,<nowiki>#</nowiki>8,cheZ,lexA③,<nowiki>#</nowiki>3-11-5(1),<nowiki>#</nowiki>23,<nowiki>#</nowiki>7(EX),<nowiki>#</nowiki>20,<nowiki>#</nowiki>7(SP))
+
*Gel extraction and Nanodrop(<nowiki>#</nowiki>6, <nowiki>#</nowiki>27, <nowiki>#</nowiki>8, cheZ, lexA③, <nowiki>#</nowiki>3-11-5(1), <nowiki>#</nowiki>23, <nowiki>#</nowiki>7_EX, <nowiki>#</nowiki>20, <nowiki>#</nowiki>7_SP)
*Ligation(<nowiki>#</nowiki>27-psB1A2, <nowiki>#</nowiki>3-27, <nowiki>#</nowiki>20-28, <nowiki>#</nowiki>28-psB1A2, <nowiki>#</nowiki>3-29, <nowiki>#</nowiki>29-psB1A2, <nowiki>#</nowiki>31-psB1A2, <nowiki>#</nowiki>3-<nowiki>#</nowiki>23, <nowiki>#</nowiki>1-<nowiki>#</nowiki>3-11-5(1), <nowiki>#</nowiki>2-<nowiki>#</nowiki>3-11-5(1), <nowiki>#</nowiki>24-<nowiki>#</nowiki>3-11-5(1), <nowiki>#</nowiki>3-<nowiki>#</nowiki>30)
*Ligation(<nowiki>#</nowiki>27-psB1A2, <nowiki>#</nowiki>3-27, <nowiki>#</nowiki>20-28, <nowiki>#</nowiki>28-psB1A2, <nowiki>#</nowiki>3-29, <nowiki>#</nowiki>29-psB1A2, <nowiki>#</nowiki>31-psB1A2, <nowiki>#</nowiki>3-<nowiki>#</nowiki>23, <nowiki>#</nowiki>1-<nowiki>#</nowiki>3-11-5(1), <nowiki>#</nowiki>2-<nowiki>#</nowiki>3-11-5(1), <nowiki>#</nowiki>24-<nowiki>#</nowiki>3-11-5(1), <nowiki>#</nowiki>3-<nowiki>#</nowiki>30)
*Making LB plate
*Making LB plate
Line 398: Line 409:
*Colony PCR(<nowiki>#</nowiki>27-pSB1A2, <nowiki>#</nowiki>3-27, <nowiki>#</nowiki>20-28, <nowiki>#</nowiki>28-psB1A2, <nowiki>#</nowiki>3-29, <nowiki>#</nowiki>29-psB1A2, <nowiki>#</nowiki>31-pSB1A2, <nowiki>#</nowiki>3-<nowiki>#</nowiki>23, <nowiki>#</nowiki>1-<nowiki>#</nowiki>3-11-5(1), <nowiki>#</nowiki>2-<nowiki>#</nowiki>3-11-5(1), <nowiki>#</nowiki>24-<nowiki>#</nowiki>3-11-5(1), <nowiki>#</nowiki>3-<nowiki>#</nowiki>30）)
*Colony PCR(<nowiki>#</nowiki>27-pSB1A2, <nowiki>#</nowiki>3-27, <nowiki>#</nowiki>20-28, <nowiki>#</nowiki>28-psB1A2, <nowiki>#</nowiki>3-29, <nowiki>#</nowiki>29-psB1A2, <nowiki>#</nowiki>31-pSB1A2, <nowiki>#</nowiki>3-<nowiki>#</nowiki>23, <nowiki>#</nowiki>1-<nowiki>#</nowiki>3-11-5(1), <nowiki>#</nowiki>2-<nowiki>#</nowiki>3-11-5(1), <nowiki>#</nowiki>24-<nowiki>#</nowiki>3-11-5(1), <nowiki>#</nowiki>3-<nowiki>#</nowiki>30）)
*Cloning(lexA, cheZ)
*Cloning(lexA, cheZ)
-
*Digest(<nowiki>#</nowiki>6 EScut, <nowiki>#</nowiki>7 EXcut, <nowiki>#</nowiki>1,<nowiki>#</nowiki>2,<nowiki>#</nowiki>3,<nowiki>#</nowiki>7,<nowiki>#</nowiki>20 SPcut, <nowiki>#</nowiki>23 XPcut）
+
*Digest
 +
**EScut : <nowiki>#</nowiki>6
 +
**EXcut : <nowiki>#</nowiki>7
 +
**SPcut : <nowiki>#</nowiki>1, <nowiki>#</nowiki>2, <nowiki>#</nowiki>3, <nowiki>#</nowiki>7, <nowiki>#</nowiki>20
 +
**XPcut : <nowiki>#</nowiki>23
*Defrost Primer
*Defrost Primer
=='11/08/11(Thu)==
=='11/08/11(Thu)==
-
*Gel extraction(<nowiki>#</nowiki>30,<nowiki>#</nowiki>14-5(insert,vector),<nowiki>#</nowiki>5,<nowiki>#</nowiki>3-11-5,cheZ,lexA)
+
*Gel extraction(<nowiki>#</nowiki>30, <nowiki>#</nowiki>14-5(insert,vector), <nowiki>#</nowiki>5, <nowiki>#</nowiki>3-11-5, cheZ, lexA)
*Cloning(sulAp, uvrAp)
*Cloning(sulAp, uvrAp)
-
*Miniprep(<nowiki>#</nowiki>3, <nowiki>#</nowiki>20-28,<nowiki>#</nowiki>31,<nowiki>#</nowiki>24-3-11-5,<nowiki>#</nowiki>3-30)
+
*Miniprep(<nowiki>#</nowiki>3, <nowiki>#</nowiki>20-28, <nowiki>#</nowiki>31, <nowiki>#</nowiki>24-3-11-5, <nowiki>#</nowiki>3-30)
=='11/08/12(Fri)==
=='11/08/12(Fri)==
*Colony PCR(<nowiki>#</nowiki>3-14-5(2), <nowiki>#</nowiki>6-5, <nowiki>#</nowiki>3-23)
*Colony PCR(<nowiki>#</nowiki>3-14-5(2), <nowiki>#</nowiki>6-5, <nowiki>#</nowiki>3-23)
-
*Cloning（nested-PCR）(uvrAp, sulAp)
+
*Cloning(nested-PCR)(uvrAp, sulAp)
-
*Check Electrophoresis（8/11 PCR product：<nowiki>#</nowiki>27, <nowiki>#</nowiki>28, Miniprep product：<nowiki>#</nowiki>3-14-5(2),<nowiki>#</nowiki>5）
+
*Check Electrophoresis
 +
**8/11 PCR product：<nowiki>#</nowiki>27, <nowiki>#</nowiki>28
 +
**Miniprep product：<nowiki>#</nowiki>3-14-5(2), <nowiki>#</nowiki>5
*Digest（<nowiki>#</nowiki>5 EXcut, <nowiki>#</nowiki>14 EScut）
*Digest（<nowiki>#</nowiki>5 EXcut, <nowiki>#</nowiki>14 EScut）
-
*Gel extraction(8/11 digest product : <nowiki>#</nowiki>5, <nowiki>#</nowiki>14-5, <nowiki>#</nowiki>3-30, cheZ, lexA, 8/11 PCR product : <nowiki>#</nowiki>27, <nowiki>#</nowiki>28 8/12 nested-PCR product : sulAp, uvrAp)
+
*Gel extraction
 +
**8/11 digest product : <nowiki>#</nowiki>5, <nowiki>#</nowiki>14-5, <nowiki>#</nowiki>3-30, cheZ, lexA
 +
**8/11 PCR product : <nowiki>#</nowiki>27, <nowiki>#</nowiki>28
 +
**8/12 nested-PCR product : sulAp, uvrAp
*Colony PCR (re) (<nowiki>#</nowiki>13-14-5)
*Colony PCR (re) (<nowiki>#</nowiki>13-14-5)
*Miniprep(ligation product : <nowiki>#</nowiki>6-5, <nowiki>#</nowiki>3-23)
*Miniprep(ligation product : <nowiki>#</nowiki>6-5, <nowiki>#</nowiki>3-23)
-
*Cloning(hotstart-PCR: uvrBp & cheZ)
+
*Cloning(hotstart-PCR: uvrBp , cheZ)
-
*Digest(XPcut:<nowiki>#</nowiki>27, <nowiki>#</nowiki>28, <nowiki>#</nowiki>6-5 EScut: sulAp, uvrAp, <nowiki>#</nowiki>3-23)
+
*Digest
 +
**XPcut:<nowiki>#</nowiki>27, <nowiki>#</nowiki>28, <nowiki>#</nowiki>6-5
 +
**EScut: sulAp, uvrAp, <nowiki>#</nowiki>3-23
=='11/08/15(Mon)==
=='11/08/15(Mon)==
-
*Gel extraction(from previous digest products(8/12):<nowiki>#</nowiki>27, <nowiki>#</nowiki>28, <nowiki>#</nowiki>33, <nowiki>#</nowiki>34, %, <nowiki>#</nowiki>4, <nowiki>#</nowiki>3-23)
+
*Gel extraction(from previous digest products(8/12) : <nowiki>#</nowiki>27, <nowiki>#</nowiki>28, <nowiki>#</nowiki>33, <nowiki>#</nowiki>34, <nowiki>#</nowiki>4, <nowiki>#</nowiki>3-23)
-
*Digest(XPcut: cheZ, uvrBp(PCR products) EScut: <nowiki>#</nowiki>3-11-5 to obtain pSB1AK3 EScut)
+
*Digest
 +
**XPcut: cheZ, uvrBp(PCR products)  
 +
**EScut: <nowiki>#</nowiki>3-11-5 (to obtain pSB1AK3 EScut)
*Assay of Cell lysis construction(pLuc-Lysis device) inducted by IPTG
*Assay of Cell lysis construction(pLuc-Lysis device) inducted by IPTG
*Making Lysis buffer, Agarose gel
*Making Lysis buffer, Agarose gel
*Gel extraction(from digest products XPcut: cheZ, uvrBp(PCR products) EScut: <nowiki>#</nowiki>3-11-5 to obtain pSB1AK3 EScut)
*Gel extraction(from digest products XPcut: cheZ, uvrBp(PCR products) EScut: <nowiki>#</nowiki>3-11-5 to obtain pSB1AK3 EScut)
*Ligation(<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5, <nowiki>#</nowiki>24-<nowiki>#</nowiki>3-17-5, <nowiki>#</nowiki>24-<nowiki>#</nowiki>3-11-5, <nowiki>#</nowiki>20-<nowiki>#</nowiki>28, <nowiki>#</nowiki>3-<nowiki>#</nowiki>27, <nowiki>#</nowiki>3-<nowiki>#</nowiki>29, <nowiki>#</nowiki>3-23-<nowiki>#</nowiki>5, <nowiki>#</nowiki>33-pSB1AK3, <nowiki>#</nowiki>34--pSB1AK3, <nowiki>#</nowiki>35--pSB1AK3)
*Ligation(<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5, <nowiki>#</nowiki>24-<nowiki>#</nowiki>3-17-5, <nowiki>#</nowiki>24-<nowiki>#</nowiki>3-11-5, <nowiki>#</nowiki>20-<nowiki>#</nowiki>28, <nowiki>#</nowiki>3-<nowiki>#</nowiki>27, <nowiki>#</nowiki>3-<nowiki>#</nowiki>29, <nowiki>#</nowiki>3-23-<nowiki>#</nowiki>5, <nowiki>#</nowiki>33-pSB1AK3, <nowiki>#</nowiki>34--pSB1AK3, <nowiki>#</nowiki>35--pSB1AK3)
-
*Transformation(ligation products:WT, <nowiki>#</nowiki>2-9:cheZ-, <nowiki>#</nowiki>14, <nowiki>#</nowiki>17) -> 37 incubation O.N.
+
*Transformation(ligation products:WT, <nowiki>#</nowiki>2-9:cheZ-, <nowiki>#</nowiki>14, <nowiki>#</nowiki>17) -> 37 incubation
=='11/08/16(Tue)==
=='11/08/16(Tue)==
-
*Colony PCR(1:<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5, 2:<nowiki>#</nowiki>24-<nowiki>#</nowiki>3-17-5, 4:<nowiki>#</nowiki>20-<nowiki>#</nowiki>28, 5:<nowiki>#</nowiki>3-<nowiki>#</nowiki>27, 6:<nowiki>#</nowiki>3-<nowiki>#</nowiki>29, 7:<nowiki>#</nowiki>3-23-<nowiki>#</nowiki>5)
+
*Colony PCR(<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5, <nowiki>#</nowiki>24-<nowiki>#</nowiki>3-17-5, <nowiki>#</nowiki>20-<nowiki>#</nowiki>28, <nowiki>#</nowiki>3-<nowiki>#</nowiki>27, <nowiki>#</nowiki>3-<nowiki>#</nowiki>29, <nowiki>#</nowiki>3-23-<nowiki>#</nowiki>5)
-
*Culture (4:<nowiki>#</nowiki>20-<nowiki>#</nowiki>28, 14:<nowiki>#</nowiki>5, 16:<nowiki>#</nowiki>14, 17:<nowiki>#</nowiki>17, 18:<nowiki>#</nowiki>2-9, <nowiki>#</nowiki>2-4, <nowiki>#</nowiki>2-3-17-5, <nowiki>#</nowiki>1, <nowiki>#</nowiki>2)
+
*Culture (<nowiki>#</nowiki>20-<nowiki>#</nowiki>28, <nowiki>#</nowiki>5, <nowiki>#</nowiki>14, <nowiki>#</nowiki>17, <nowiki>#</nowiki>2-9, <nowiki>#</nowiki>2-4, <nowiki>#</nowiki>2-3-17-5, <nowiki>#</nowiki>1, <nowiki>#</nowiki>2)
-
*Incubation (3:<nowiki>#</nowiki>24-<nowiki>#</nowiki>3-11-5, 8:<nowiki>#</nowiki>33-pSB1AK3, 9:<nowiki>#</nowiki>34-pSB1AK3, 10:<nowiki>#</nowiki>35-pSB1AK3)
+
*Incubation (<nowiki>#</nowiki>24-<nowiki>#</nowiki>3-11-5, <nowiki>#</nowiki>33-pSB1AK3, <nowiki>#</nowiki>34-pSB1AK3, <nowiki>#</nowiki>35-pSB1AK3)
*Colony PCR(additional)(previously incubated ligation products: 3, 8, 10)
*Colony PCR(additional)(previously incubated ligation products: 3, 8, 10)
*hotstart PCR(sulAp, uvrAp, uvrBp)
*hotstart PCR(sulAp, uvrAp, uvrBp)
-
*Digest(SPcut: <nowiki>#</nowiki>3, Ecut:<nowiki>#</nowiki>3(control)) go to 37 incubation(12:30
+
*Digest(SPcut: <nowiki>#</nowiki>3, Ecut:<nowiki>#</nowiki>3(control)) go to 37 incubation
-
*Ligation retry(1:<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5, 5:<nowiki>#</nowiki>3-<nowiki>#</nowiki>27, 6:<nowiki>#</nowiki>3-<nowiki>#</nowiki>29, N.C.:<nowiki>#</nowiki>3)
+
*Ligation retry(<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5, <nowiki>#</nowiki>3-<nowiki>#</nowiki>27, <nowiki>#</nowiki>3-<nowiki>#</nowiki>29, Negative control :<nowiki>#</nowiki>3)
-
*Making Frozen stock(<nowiki>#</nowiki>14、<nowiki>#</nowiki>17)
+
*Frozen stock(<nowiki>#</nowiki>14, <nowiki>#</nowiki>17)
-
*Culture test(<nowiki>#</nowiki>14 on 0.3%Agar.(km))(17:10
+
*Culture test(<nowiki>#</nowiki>14 on 0.3%Agar.(km))
-
*Ethanol precipitation: 29.9ng/ul * 1000ul (!)
+
*Ethanol precipitation: 29.9ng/ul * 1000ul
-
*Miniprep(Ligation products: <nowiki>#</nowiki>2-3-17-5、<nowiki>#</nowiki>14、<nowiki>#</nowiki>17、<nowiki>#</nowiki>1、<nowiki>#</nowiki>2)
+
*Miniprep(Ligation products : <nowiki>#</nowiki>2-3-17-5, <nowiki>#</nowiki>14, <nowiki>#</nowiki>17, <nowiki>#</nowiki>1, <nowiki>#</nowiki>2)
*Electrophoresis(Colony PCR products: 3, 8, 10)
*Electrophoresis(Colony PCR products: 3, 8, 10)
*Waking from frozen stock(<nowiki>#</nowiki>3, CheZ-, WT)
*Waking from frozen stock(<nowiki>#</nowiki>3, CheZ-, WT)
=='11/08/17(Wed)==
=='11/08/17(Wed)==
-
*Hotstart-PCR (Change conditions a little )
+
*Hotstart-PCR (Change conditions a little)
-
**from Genome
+
**From Genome
-
**from nested-region
+
**From nested-region
-
**from parts
+
**From parts
-
*Making Competent Cell, Sob medium
+
*Making Competent Cell, SOB medium
*Ethanol precipitation(WT: 40ng/ul)
*Ethanol precipitation(WT: 40ng/ul)
-
*Culture from master-plate(<nowiki>#</nowiki>6-5, <nowiki>#</nowiki>20-28, <nowiki>#</nowiki>3-27, <nowiki>#</nowiki>33, <nowiki>#</nowiki>24-3-11-5 <nowiki>#</nowiki>24-3-17-5) -> 21:00 Miniprep
+
*Culture from master-plate(<nowiki>#</nowiki>6-5, <nowiki>#</nowiki>20-28, <nowiki>#</nowiki>3-27, <nowiki>#</nowiki>33, <nowiki>#</nowiki>24-3-11-5 <nowiki>#</nowiki>24-3-17-5) -> Miniprep
*Assay(IPTG induced cell lysis device
*Assay(IPTG induced cell lysis device
*Gel extraction(SPcut:<nowiki>#</nowiki>3)
*Gel extraction(SPcut:<nowiki>#</nowiki>3)
*Culture from Frozen stock(<nowiki>#</nowiki>1, <nowiki>#</nowiki>2, <nowiki>#</nowiki>2-3-17-5)
*Culture from Frozen stock(<nowiki>#</nowiki>1, <nowiki>#</nowiki>2, <nowiki>#</nowiki>2-3-17-5)
-
*Electrophresis check!!!(Loading only DNA size markers)
+
*Electrophresis check(Loading only DNA size markers)
*Miniprep(<nowiki>#</nowiki>3, <nowiki>#</nowiki>6-5, <nowiki>#</nowiki>20-28, <nowiki>#</nowiki>3-27, <nowiki>#</nowiki>33, <nowiki>#</nowiki>24-3-11-5 <nowiki>#</nowiki>24-3-17-5)
*Miniprep(<nowiki>#</nowiki>3, <nowiki>#</nowiki>6-5, <nowiki>#</nowiki>20-28, <nowiki>#</nowiki>3-27, <nowiki>#</nowiki>33, <nowiki>#</nowiki>24-3-11-5 <nowiki>#</nowiki>24-3-17-5)
=='11/08/18(Thu)==
=='11/08/18(Thu)==
-
*Digest(8/17<nowiki>#</nowiki>3,<nowiki>#</nowiki>33 SPcut, <nowiki>#</nowiki>14-5 XPcut, <nowiki>#</nowiki>33 Ecut, <nowiki>#</nowiki>3-27,<nowiki>#</nowiki>3-30,<nowiki>#</nowiki>20-28 EScut, pSB1K3 EPcut)
+
*Digest(8/17<nowiki>#</nowiki>3, <nowiki>#</nowiki>33 SPcut, <nowiki>#</nowiki>14-5 XPcut, <nowiki>#</nowiki>33 Ecut, <nowiki>#</nowiki>3-27,<nowiki>#</nowiki>3-30,<nowiki>#</nowiki>20-28 EScut, pSB1K3 EPcut)
-
*Hotstart-PCR (Change conditions a little )
+
*Hotstart-PCR (Change conditions a little)
**From Genome :  uvrBp, cheZ
**From Genome :  uvrBp, cheZ
**From nested-region : uvrA
**From nested-region : uvrA
*Culture from 0809 master-plate ⑪ (<nowiki>#</nowiki>2-3-11-5) → Miniprep.
*Culture from 0809 master-plate ⑪ (<nowiki>#</nowiki>2-3-11-5) → Miniprep.
*Making LB plate & tube with various agarose conc. (plate: 0.5, 0.7, 0.9%×2 each, tube: 0.5,1.0,1.5,2.0,3.0%×2 each)(selection: Kanamycin)
*Making LB plate & tube with various agarose conc. (plate: 0.5, 0.7, 0.9%×2 each, tube: 0.5,1.0,1.5,2.0,3.0%×2 each)(selection: Kanamycin)
-
*Gel extraction(8/17<nowiki>#</nowiki>3,<nowiki>#</nowiki>33 SPcut, <nowiki>#</nowiki>14-5 XPcut, <nowiki>#</nowiki>33 Ecut, <nowiki>#</nowiki>3-27 EScut, pSB1K3 EPcut)
+
*Gel extraction(8/17<nowiki>#</nowiki>3_SP,<nowiki>#</nowiki>33_SP, <nowiki>#</nowiki>14-5_XP, <nowiki>#</nowiki>33_E, <nowiki>#</nowiki>3-27_ES, pSB1K3 _EP)
*Miniprep(<nowiki>#</nowiki>1, <nowiki>#</nowiki>2, <nowiki>#</nowiki>2-3-11-5, <nowiki>#</nowiki>2-3-17-5)
*Miniprep(<nowiki>#</nowiki>1, <nowiki>#</nowiki>2, <nowiki>#</nowiki>2-3-11-5, <nowiki>#</nowiki>2-3-17-5)
-
*Digest(<nowiki>#</nowiki>1,<nowiki>#</nowiki>2 SPcut, <nowiki>#</nowiki>2-3-17-5 XPcut, <nowiki>#</nowiki>2-3-11-5 SPcut(check),EPcut(selection), <nowiki>#</nowiki>3-30,<nowiki>#</nowiki>20-28 EScut, 0817 Gel extracted PCR product (uvrBp) EScut, 0818PCR products (uvrAp, uvrBp, cheZ) ES&XPcut) →O.N.
+
*Digest(<nowiki>#</nowiki>1,<nowiki>#</nowiki>2 SPcut, <nowiki>#</nowiki>2-3-17-5 XPcut, <nowiki>#</nowiki>2-3-11-5 SPcut(check), EPcut(selection), <nowiki>#</nowiki>3-30, <nowiki>#</nowiki>20-28 EScut, 0817 Gel extracted PCR product (uvrBp) EScut, 0818PCR products (uvrAp, uvrBp, cheZ) ES&XPcut)
*Ligation (8/17<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5, 8/17<nowiki>#</nowiki>3-8/15<nowiki>#</nowiki>29, <nowiki>#</nowiki>33-<nowiki>#</nowiki>3-11-5, 8/15<nowiki>#</nowiki>34-pSB1AK3, <nowiki>#</nowiki>3-27-<nowiki>#</nowiki>5, <nowiki>#</nowiki>20-28-pSB1AK3, <nowiki>#</nowiki>28-pSB1AK3)
*Ligation (8/17<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5, 8/17<nowiki>#</nowiki>3-8/15<nowiki>#</nowiki>29, <nowiki>#</nowiki>33-<nowiki>#</nowiki>3-11-5, 8/15<nowiki>#</nowiki>34-pSB1AK3, <nowiki>#</nowiki>3-27-<nowiki>#</nowiki>5, <nowiki>#</nowiki>20-28-pSB1AK3, <nowiki>#</nowiki>28-pSB1AK3)
-
*Culture for Frozen stock in eppendolf tube (O.N. in 37℃ incubator)
+
*Culture for frozen stock in eppendolf tube
=='11/08/19(Fri)==
=='11/08/19(Fri)==
*ColonyPCR(from ligation products)(<nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>3-29, <nowiki>#</nowiki>33-3-11-5, <nowiki>#</nowiki>34-pSB1AK3, <nowiki>#</nowiki>3-27-5)
*ColonyPCR(from ligation products)(<nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>3-29, <nowiki>#</nowiki>33-3-11-5, <nowiki>#</nowiki>34-pSB1AK3, <nowiki>#</nowiki>3-27-5)
-
*Gel extraction(08/18's digest products:<nowiki>#</nowiki>1, <nowiki>#</nowiki>2, <nowiki>#</nowiki>2-3-11-5 SPcut, <nowiki>#</nowiki>2-3-17-5, cheZ XPcut, <nowiki>#</nowiki>2-3-11-5 EPcut, 8/18uvrBp, 8/17uvrBp, uvrAp EScut)
+
*Gel extraction(08/18's digest products:<nowiki>#</nowiki>1_SP, <nowiki>#</nowiki>2_SP, <nowiki>#</nowiki>2-3-11-5_SP, <nowiki>#</nowiki>2-3-17-5_XP, cheZ_XP, <nowiki>#</nowiki>2-3-11-5_EP, 8/18uvrBp_ES, 8/17uvrBp_ES, uvrAp_ES)
*Colony PCR retry(<nowiki>#</nowiki>3-29, <nowiki>#</nowiki>34-pSB1AK3)
*Colony PCR retry(<nowiki>#</nowiki>3-29, <nowiki>#</nowiki>34-pSB1AK3)
-
*Making Frozen Stock
+
*Frozen Stock
-
*Check the list of frozen stock(there were too many mistakes)
+
*Check the list of frozen stock
*Assay of recAp from biobrick parts using RFP construct & transilluminator
*Assay of recAp from biobrick parts using RFP construct & transilluminator
*Electrophoresis(<nowiki>#</nowiki>3, <nowiki>#</nowiki>3-27, <nowiki>#</nowiki>3-30, <nowiki>#</nowiki>6-5, <nowiki>#</nowiki>7, <nowiki>#</nowiki>20-28, <nowiki>#</nowiki>24-3-11-5, <nowiki>#</nowiki>24-3-17-5, <nowiki>#</nowiki>33)
*Electrophoresis(<nowiki>#</nowiki>3, <nowiki>#</nowiki>3-27, <nowiki>#</nowiki>3-30, <nowiki>#</nowiki>6-5, <nowiki>#</nowiki>7, <nowiki>#</nowiki>20-28, <nowiki>#</nowiki>24-3-11-5, <nowiki>#</nowiki>24-3-17-5, <nowiki>#</nowiki>33)
=='11/08/22(Mon)==
=='11/08/22(Mon)==
-
*Making SOB medium(1000ml)
+
*Making SOB medium
*Culture for Miniprep
*Culture for Miniprep
**From frozen stock(<nowiki>#</nowiki>1, <nowiki>#</nowiki>2, <nowiki>#</nowiki>7, <nowiki>#</nowiki>2-3-17-5(2), <nowiki>#</nowiki>3-30)
**From frozen stock(<nowiki>#</nowiki>1, <nowiki>#</nowiki>2, <nowiki>#</nowiki>7, <nowiki>#</nowiki>2-3-17-5(2), <nowiki>#</nowiki>3-30)
Line 511: Line 535:
**<nowiki>#</nowiki>27 (82.1 ng/ul, total = 30 ul) -> XP cut
**<nowiki>#</nowiki>27 (82.1 ng/ul, total = 30 ul) -> XP cut
**<nowiki>#</nowiki>28 (104.4 ng/ul, total = 30 ul) -> XP cut
**<nowiki>#</nowiki>28 (104.4 ng/ul, total = 30 ul) -> XP cut
-
*Making plate and amp stock　→4℃
+
*Making plate and amp stock　->4℃
**amp 32
**amp 32
**km 11 (km conc. = 3/4 * that it was)
**km 11 (km conc. = 3/4 * that it was)
Line 520: Line 544:
**Coelenterazine Solution : 100ul * 10 at -20℃
**Coelenterazine Solution : 100ul * 10 at -20℃
**Lysis buffer : 500ul * 10 at -20℃
**Lysis buffer : 500ul * 10 at -20℃
-
*Ligation(<nowiki>#</nowiki>3-30-<nowiki>#</nowiki>5, <nowiki>#</nowiki>3-11-5-<nowiki>#</nowiki>34, <nowiki>#</nowiki>30-<nowiki>#</nowiki>3, <nowiki>#</nowiki>28-<nowiki>#</nowiki>20, <nowiki>#</nowiki>35-pSB1AK3, <nowiki>#</nowiki>2-3-11-5-pSB1K3, <nowiki>#</nowiki>14-5-<nowiki>#</nowiki>3 and N.C)
+
*Ligation(<nowiki>#</nowiki>3-30-<nowiki>#</nowiki>5, <nowiki>#</nowiki>3-11-5-<nowiki>#</nowiki>34, <nowiki>#</nowiki>30-<nowiki>#</nowiki>3, <nowiki>#</nowiki>28-<nowiki>#</nowiki>20, <nowiki>#</nowiki>35-pSB1AK3, <nowiki>#</nowiki>2-3-11-5-pSB1K3, <nowiki>#</nowiki>14-5-<nowiki>#</nowiki>3 and Negative control)
=='11/08/25(Thu)==
=='11/08/25(Thu)==
*Renilla luciferase assay (with <nowiki>#</nowiki>2-3-17-5 culture)
*Renilla luciferase assay (with <nowiki>#</nowiki>2-3-17-5 culture)
-
*Gel extraction (<nowiki>#</nowiki>27,<nowiki>#</nowiki>28 XPcut)
+
*Gel extraction (<nowiki>#</nowiki>27, <nowiki>#</nowiki>28 XPcut)
*Ligation (0824 retry & <nowiki>#</nowiki>20-<nowiki>#</nowiki>28, <nowiki>#</nowiki>3-<nowiki>#</nowiki>27)
*Ligation (0824 retry & <nowiki>#</nowiki>20-<nowiki>#</nowiki>28, <nowiki>#</nowiki>3-<nowiki>#</nowiki>27)
-
*Transformation (Ligation products into WT competent cells & <nowiki>#</nowiki>14-5 miniprep product into WT cell (line-drawing spread) & <nowiki>#</nowiki>33 miniprep product into ΔcheZ cell (line-drawing spread))  
+
*Transformation (Ligation products into WT competent cells & <nowiki>#</nowiki>14-5 miniprep product into WT cell (line-drawing spread), <nowiki>#</nowiki>33 miniprep product into ΔcheZ cell (line-drawing spread))  
*Digest (<nowiki>#</nowiki>14 XPcut)
*Digest (<nowiki>#</nowiki>14 XPcut)
-
*Passage（<nowiki>#</nowiki>2-3-17-5, WT, ΔcheZ, WT in eppen, ΔcheZ in eppen)
+
*Passage(<nowiki>#</nowiki>2-3-17-5, WT, ΔcheZ, WT in eppen, ΔcheZ in eppen)
=='11/08/26(Fri)==
=='11/08/26(Fri)==
*Renilla luciferase assay (with <nowiki>#</nowiki>2-3-17-5 culture)
*Renilla luciferase assay (with <nowiki>#</nowiki>2-3-17-5 culture)
*Colony PCR (<nowiki>#</nowiki>33 Miniprep transformations)
*Colony PCR (<nowiki>#</nowiki>33 Miniprep transformations)
-
*Gel extraction (<nowiki>#</nowiki>14 XPcut)
+
*Gel extraction (<nowiki>#</nowiki>14_XP)
-
*Ligation (retry) (using today's competent cells & 0802 ΔcheZ cells)
+
*Ligation (retry) (using today's competent cells, 0802 ΔcheZ cells)
*Cloning (cheZ)
*Cloning (cheZ)
*Digest (<nowiki>#</nowiki>5 EXcut, cheZ PCR products XPcut)
*Digest (<nowiki>#</nowiki>5 EXcut, cheZ PCR products XPcut)
-
*Passage in a shaker（<nowiki>#</nowiki>2-3-17-5, WT, ΔcheZ）
+
*Passage in a shaker(<nowiki>#</nowiki>2-3-17-5, WT, ΔcheZ)
*Making competent cell, LB broth
*Making competent cell, LB broth
*Contamination check (0817 comp., 0802 comp., 0802 ⊿cheZ comp. spread on LB (amp) plates)
*Contamination check (0817 comp., 0802 comp., 0802 ⊿cheZ comp. spread on LB (amp) plates)
Line 543: Line 567:
=='11/08/27(Sat)==
=='11/08/27(Sat)==
*Ligation & Transformation (0826 ①②③⑤⑧⑨) (into Nippon Gene comp.)
*Ligation & Transformation (0826 ①②③⑤⑧⑨) (into Nippon Gene comp.)
-
*Contamination check (WT FS, ΔcheZ FS)
+
*Contamination check (WT FS, ΔcheZ Frozen stock)
-
*Waking from FS (WT, ΔcheZ)
+
*Waking from Frozen stock(WT, ΔcheZ)
=='11/08/28(Sun)==
=='11/08/28(Sun)==
*Making ΔcheZ comp., LB amp plates, TB
*Making ΔcheZ comp., LB amp plates, TB
*Colony PCR & master plate preparation -> Failure!!
*Colony PCR & master plate preparation -> Failure!!
-
*Gel extraction (<nowiki>#</nowiki>29 XPcut, <nowiki>#</nowiki>5 EXcut)
+
*Gel extraction (<nowiki>#</nowiki>29_XP, <nowiki>#</nowiki>5_EX)
-
*Miniprep & frozen stocks (<nowiki>#</nowiki>33, <nowiki>#</nowiki>14-5)
+
*Miniprep and frozen stocks (<nowiki>#</nowiki>33, <nowiki>#</nowiki>14-5)
*Digest (<nowiki>#</nowiki>20 SPcut, <nowiki>#</nowiki>14-5 XPcut)
*Digest (<nowiki>#</nowiki>20 SPcut, <nowiki>#</nowiki>14-5 XPcut)
-
*Waking from Frozen stock (WT, ⊿cheZ, <nowiki>#</nowiki>20, <nowiki>#</nowiki>14-5)
+
*Waking from frozen stock (WT, ⊿cheZ, <nowiki>#</nowiki>20, <nowiki>#</nowiki>14-5)
=='11/08/29(Mon)==
=='11/08/29(Mon)==
-
*PCR purification -> digest with XP
+
*PCR purification -> digest with XP(<nowiki>#</nowiki>27, <nowiki>#</nowiki>28)
-
**<nowiki>#</nowiki>27(251.3ng/ul),<nowiki>#</nowiki>28
+
*Miniprep(<nowiki>#</nowiki>20, <nowiki>#</nowiki>14-5)
-
*Miniprep
+
-
**<nowiki>#</nowiki>20(128.1ng/ul), <nowiki>#</nowiki>14-5
+
*Gel extraction(<nowiki>#</nowiki>14-5, <nowiki>#</nowiki>20)
*Gel extraction(<nowiki>#</nowiki>14-5, <nowiki>#</nowiki>20)
*Ligation (higher I/V ratio, into cheZ comp.)(<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5(retry), <nowiki>#</nowiki>2-3-11-5 to pSB1K3 (retry), <nowiki>#</nowiki>35-pSB1AK3, (<nowiki>#</nowiki>33,<nowiki>#</nowiki>34)-<nowiki>#</nowiki>3-17-5)
*Ligation (higher I/V ratio, into cheZ comp.)(<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5(retry), <nowiki>#</nowiki>2-3-11-5 to pSB1K3 (retry), <nowiki>#</nowiki>35-pSB1AK3, (<nowiki>#</nowiki>33,<nowiki>#</nowiki>34)-<nowiki>#</nowiki>3-17-5)
=='11/08/30(Tue)==
=='11/08/30(Tue)==
-
*Making reagents and medium
+
*Making LB amp, Aspartic acid solution (10mM), H2O2 solution (10mM)
-
**LB amp
+
*Gel extraction -> PCR was failed!(<nowiki>#</nowiki>27,<nowiki>#</nowiki>28)
-
**Aspartic acid solution (10mM)
+
-
**H2O2 solution (10mM)
+
-
*Gel extraction -> PCR was failed!
+
-
**<nowiki>#</nowiki>27,<nowiki>#</nowiki>28
+
*There was trash (from PCR) below
*There was trash (from PCR) below
*Colony PCR(<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5, <nowiki>#</nowiki>2-3-11-5 to pSB1K3, <nowiki>#</nowiki>35-pSB1AK3, (<nowiki>#</nowiki>33,<nowiki>#</nowiki>34)-<nowiki>#</nowiki>3-17-5)
*Colony PCR(<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5, <nowiki>#</nowiki>2-3-11-5 to pSB1K3, <nowiki>#</nowiki>35-pSB1AK3, (<nowiki>#</nowiki>33,<nowiki>#</nowiki>34)-<nowiki>#</nowiki>3-17-5)
Line 587: Line 605:
*Gel extraction(<nowiki>#</nowiki>20_SP, pSB1AK3_EP, <nowiki>#</nowiki>3_SP)
*Gel extraction(<nowiki>#</nowiki>20_SP, pSB1AK3_EP, <nowiki>#</nowiki>3_SP)
*PCR -> clean-up -> XPcut(<nowiki>#</nowiki>27, <nowiki>#</nowiki>28, <nowiki>#</nowiki>14-5, <nowiki>#</nowiki>3-30)
*PCR -> clean-up -> XPcut(<nowiki>#</nowiki>27, <nowiki>#</nowiki>28, <nowiki>#</nowiki>14-5, <nowiki>#</nowiki>3-30)
-
*Waking from Frozen stock(<nowiki>#</nowiki>2-9 (WT), <nowiki>#</nowiki>24-3-17-5)
+
*Waking from frozen stock(<nowiki>#</nowiki>2-9 (WT), <nowiki>#</nowiki>24-3-17-5)
*Plating from liquid culture(<nowiki>#</nowiki>2-3-17-5)
*Plating from liquid culture(<nowiki>#</nowiki>2-3-17-5)
*Ligation(<nowiki>#</nowiki>20-<nowiki>#</nowiki>3-17-5, <nowiki>#</nowiki>2-3-11-5-pSB1AK3, <nowiki>#</nowiki>3-<nowiki>#</nowiki>14, <nowiki>#</nowiki>20-<nowiki>#</nowiki>28, <nowiki>#</nowiki>3-<nowiki>#</nowiki>27, <nowiki>#</nowiki>3-<nowiki>#</nowiki>29, <nowiki>#</nowiki>35-pSB1AK3, <nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5, <nowiki>#</nowiki>3-30-<nowiki>#</nowiki>5, <nowiki>#</nowiki>33-<nowiki>#</nowiki>3-17-5 & Negative control)
*Ligation(<nowiki>#</nowiki>20-<nowiki>#</nowiki>3-17-5, <nowiki>#</nowiki>2-3-11-5-pSB1AK3, <nowiki>#</nowiki>3-<nowiki>#</nowiki>14, <nowiki>#</nowiki>20-<nowiki>#</nowiki>28, <nowiki>#</nowiki>3-<nowiki>#</nowiki>27, <nowiki>#</nowiki>3-<nowiki>#</nowiki>29, <nowiki>#</nowiki>35-pSB1AK3, <nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5, <nowiki>#</nowiki>3-30-<nowiki>#</nowiki>5, <nowiki>#</nowiki>33-<nowiki>#</nowiki>3-17-5 & Negative control)
*Transformation
*Transformation
-
**Plasmid isolation
+
**Plasmid isolation(<nowiki>#</nowiki>34-3-17-5(MP;1:9gradient))
-
**<nowiki>#</nowiki>34-3-17-5(MP;1:9gradient)
+
**Part cloning(<nowiki>#</nowiki>36, <nowiki>#</nowiki>37)
**Part cloning(<nowiki>#</nowiki>36, <nowiki>#</nowiki>37)
**Contamination check(pSB1AK3(EPcut), pSB1K3(EPcut))
**Contamination check(pSB1AK3(EPcut), pSB1K3(EPcut))
Line 601: Line 618:
*Colony PCR
*Colony PCR
**<nowiki>#</nowiki>34-3-17-5(MP), <nowiki>#</nowiki>3-14, <nowiki>#</nowiki>3-29, <nowiki>#</nowiki>35-pSB1AK3, <nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>37(from Part cloning)
**<nowiki>#</nowiki>34-3-17-5(MP), <nowiki>#</nowiki>3-14, <nowiki>#</nowiki>3-29, <nowiki>#</nowiki>35-pSB1AK3, <nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>37(from Part cloning)
-
*Gel ext.
+
*Gel extraction
-
**<nowiki>#</nowiki>27XP ,<nowiki>#</nowiki>28XP(0830); <nowiki>#</nowiki>27XP, <nowiki>#</nowiki>28XP, <nowiki>#</nowiki>3-30XP -> failed!, <nowiki>#</nowiki>14-5XP(0831)
+
**<nowiki>#</nowiki>27_XP ,<nowiki>#</nowiki>28_XP(0830); <nowiki>#</nowiki>27_XP, <nowiki>#</nowiki>28_XP, <nowiki>#</nowiki>3-30_XP -> failed!, <nowiki>#</nowiki>14-5_XP(0831)
-
*Making medium(SOB, LB amp plate)
+
*Making medium(SOB, LB amp plate), competent cells (WT & ΔcheZ)
-
*Making competent cells (WT & ΔcheZ)
+
*Miniprep(<nowiki>#</nowiki>3, <nowiki>#</nowiki>20)
*Miniprep(<nowiki>#</nowiki>3, <nowiki>#</nowiki>20)
-
*PCR & Column purification
+
*PCR and Column purification
**<nowiki>#</nowiki>3-14, <nowiki>#</nowiki>3-29, <nowiki>#</nowiki>3-14-5 (Recovery from colony PCR tubes!)
**<nowiki>#</nowiki>3-14, <nowiki>#</nowiki>3-29, <nowiki>#</nowiki>3-14-5 (Recovery from colony PCR tubes!)
*Digest
*Digest
Line 617: Line 633:
**Ligation products into Takara comp. (including N.C.)
**Ligation products into Takara comp. (including N.C.)
**Titer check(MP product <nowiki>#</nowiki>3 into 0901 WT comp., MP product <nowiki>#</nowiki>20 into 0901 ΔcheZ comp.)
**Titer check(MP product <nowiki>#</nowiki>3 into 0901 WT comp., MP product <nowiki>#</nowiki>20 into 0901 ΔcheZ comp.)
-
*Mobility assay
+
*Mobility assay(<nowiki>#</nowiki>14-5 (km resistance) WT/ΔcheZ cells)
-
**<nowiki>#</nowiki>14-5 (km resistance) WT/ΔcheZ cells
+
=='11/09/02(Fri)==
=='11/09/02(Fri)==
Line 632: Line 647:
**<nowiki>#</nowiki>20-3-17-5, <nowiki>#</nowiki>3-27, <nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>3-29 (from miniprep products)
**<nowiki>#</nowiki>20-3-17-5, <nowiki>#</nowiki>3-27, <nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>3-29 (from miniprep products)
*Digest
*Digest
-
**<nowiki>#</nowiki>3_SP, <nowiki>#</nowiki>20_SP, <nowiki>#</nowiki>37_ES, <nowiki>#</nowiki>3-14_ES, <nowiki>#</nowiki>3-29_ES, <nowiki>#</nowiki>3-14-5_XP, ΔcheZ?_E/P (from MP products)
+
**<nowiki>#</nowiki>3_SP, <nowiki>#</nowiki>20_SP, <nowiki>#</nowiki>37_ES, <nowiki>#</nowiki>3-14_ES, <nowiki>#</nowiki>3-29_ES, <nowiki>#</nowiki>3-14-5_XP, ΔcheZ_E/P (from MP products)
**<nowiki>#</nowiki>20-3-17-5_XP, <nowiki>#</nowiki>3-27_ES, <nowiki>#</nowiki>3-14-5_XP, <nowiki>#</nowiki>3-29_ES (from PCR products)
**<nowiki>#</nowiki>20-3-17-5_XP, <nowiki>#</nowiki>3-27_ES, <nowiki>#</nowiki>3-14-5_XP, <nowiki>#</nowiki>3-29_ES (from PCR products)
Line 695: Line 710:
**<nowiki>#</nowiki>33-<nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>34-<nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>36-<nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>2-<nowiki>#</nowiki>3-29-5, <nowiki>#</nowiki>20-<nowiki>#</nowiki>3-29-5, <nowiki>#</nowiki>3-<nowiki>#</nowiki>37-5, <nowiki>#</nowiki>2-3-11-5-pSB1AK3, <nowiki>#</nowiki>1-<nowiki>#</nowiki>3-17-5
**<nowiki>#</nowiki>33-<nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>34-<nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>36-<nowiki>#</nowiki>3-14-5, <nowiki>#</nowiki>2-<nowiki>#</nowiki>3-29-5, <nowiki>#</nowiki>20-<nowiki>#</nowiki>3-29-5, <nowiki>#</nowiki>3-<nowiki>#</nowiki>37-5, <nowiki>#</nowiki>2-3-11-5-pSB1AK3, <nowiki>#</nowiki>1-<nowiki>#</nowiki>3-17-5
**<nowiki>#</nowiki>33, <nowiki>#</nowiki>34, <nowiki>#</nowiki>36, <nowiki>#</nowiki>1, <nowiki>#</nowiki>2, <nowiki>#</nowiki>20, <nowiki>#</nowiki>3, pSB1AK3 (N.C.)
**<nowiki>#</nowiki>33, <nowiki>#</nowiki>34, <nowiki>#</nowiki>36, <nowiki>#</nowiki>1, <nowiki>#</nowiki>2, <nowiki>#</nowiki>20, <nowiki>#</nowiki>3, pSB1AK3 (N.C.)
-
*Ninhydrin stock solution preparation (10%, H2O : EtOH = 9:1)
+
*Ninhydrin stock solution preparation
**Asp conc. check (it took 5 min at 80C)
**Asp conc. check (it took 5 min at 80C)
*Making M9 medium(.25, .125% Agar.)
*Making M9 medium(.25, .125% Agar.)
Line 783: Line 798:
Note: <nowiki>#</nowiki>36-3-14-5-2-3-17-5-<nowiki>#</nowiki>20-3-37-5-pSB1A3 PCR amplification doesn't work. Seems failed extension...
Note: <nowiki>#</nowiki>36-3-14-5-2-3-17-5-<nowiki>#</nowiki>20-3-37-5-pSB1A3 PCR amplification doesn't work. Seems failed extension...
*PCR amplification
*PCR amplification
-
**<nowiki>#</nowiki>20-3-37-5 from Master plate
+
**From master plate : <nowiki>#</nowiki>20-3-37-5
-
**<nowiki>#</nowiki>36-3-14-5-2-3-17-5 from 0911 MP product
+
**From 09/11 Miniprep product : <nowiki>#</nowiki>36-3-14-5-2-3-17-5
Note: Only one <nowiki>#</nowiki>20-3-37-5 colony is NOT RED.
Note: Only one <nowiki>#</nowiki>20-3-37-5 colony is NOT RED.
-
*Miniprep.
+
*Miniprep(<nowiki>#</nowiki>20, <nowiki>#</nowiki>29 (pSB1AK3), <nowiki>#</nowiki>2-3-17-5, <nowiki>#</nowiki>2-3-37-5, BBa_J04450 (pSB1C3))
-
**<nowiki>#</nowiki>20
+
-
**<nowiki>#</nowiki>29 (pSB1AK3)
+
-
**<nowiki>#</nowiki>2-3-17-5
+
-
**<nowiki>#</nowiki>2-3-37-5
+
-
**BBa_J04450 (pSB1C3)
+
*Digest
*Digest
-
**<nowiki>#</nowiki>36-3-14-5-2-3-17-5_ES (from PCR)
+
**From PCR : <nowiki>#</nowiki>36-3-14-5-2-3-17-5_ES, <nowiki>#</nowiki>20-3-37-5_XP
-
**<nowiki>#</nowiki>20-3-37-5_XP (from PCR)
+
**pSB1A3_EP, pSB1C3_EP
-
**pSB1A3_EP
+
**From Miniprep : <nowiki>#</nowiki>20-3-37-5_XP
-
**pSB1C3_EP
+
**J04450_EP, *<nowiki>#</nowiki>2-9_XP, <nowiki>#</nowiki>20-3-29-5_ES
-
**<nowiki>#</nowiki>20-3-37-5_XP (from MP) -> ''O/N''
+
-
**J04450_EP -> ''O/N''
+
-
**<nowiki>#</nowiki>2-9_XP -> ''O/N''
+
-
**<nowiki>#</nowiki>20-3-29-5_ES -> ''O/N''
+
*Gel extraction
*Gel extraction
-
**<nowiki>#</nowiki>20_SP
+
**<nowiki>#</nowiki>20_SP, <nowiki>#</nowiki>36-3-14-5_SP
-
**<nowiki>#</nowiki>36-3-14-5_SP
+
**From OverNight digests : <nowiki>#</nowiki>2-3-17-5_EX
-
**<nowiki>#</nowiki>2-3-17-5_EX (from O/N digests)
+
**<nowiki>#</nowiki>36-3-14-5-2-3-17-5_ES
**<nowiki>#</nowiki>36-3-14-5-2-3-17-5_ES
-
**<nowiki>#</nowiki>20-3-37-5_XP (from today's PCR digests)
+
**From today's PCR digests : <nowiki>#</nowiki>20-3-37-5_XP
-
*Culture for Miniprep.
+
*Culture for Miniprep(<nowiki>#</nowiki>20-3-37-5 from 0911 master plate)
-
**<nowiki>#</nowiki>20-3-37-5 from 0911 master plate -> ''O/N''
+
*Dual Luciferase Assay(<nowiki>#</nowiki>20-3-14-5-2-3-17-5, <nowiki>#</nowiki>1-3-14-5-2-3-17-5, <nowiki>#</nowiki>36-3-14-5-2-3-17-5-20-3-37-5-pSB1A3)
-
*Dual Luciferase Assay
+
*Ligation
-
**<nowiki>#</nowiki>20-3-14-5-2-3-17-5
+
-
**<nowiki>#</nowiki>1-3-14-5-2-3-17-5
+
-
**<nowiki>#</nowiki>36-3-14-5-2-3-17-5-20-3-37-5-pSB1A3  
+
-
*Ligation -> ''O/N''
+
**<nowiki>#</nowiki>36-3-14-5-2-3-17-5_ES-<nowiki>#</nowiki>20-3-37-5_XP-pSB1A3_EP
**<nowiki>#</nowiki>36-3-14-5-2-3-17-5_ES-<nowiki>#</nowiki>20-3-37-5_XP-pSB1A3_EP
**<nowiki>#</nowiki>36-3-14-5-2-3-17-5_ES-<nowiki>#</nowiki>20-3-37-5_XP-pSB1C3_EP
**<nowiki>#</nowiki>36-3-14-5-2-3-17-5_ES-<nowiki>#</nowiki>20-3-37-5_XP-pSB1C3_EP

Revision as of 03:52, 15 September 2011

Parts List

Table 1. List of iGEM parts
Number Part Content Plasmid Length (bp)
#1 J23119 c.Promoter (Strong) pSB1A2 35
#2 J23118 c.Promoter (Medium) BBa_J61002 35
#3 B0032 RBS pSB1A2 13
#5 B0014 d.Ter pSB1AK3 95
#9 E0240 RBS-GFP-d.Ter pSB1A2 876
#10 E0040 GFP pSB1A2 681
#11 E1010 RFP pSB2K3 723
#14 I712019 fLuc pSB1AK8 1653
#17 J52008 rLuc pSB1AK3 936
#20 R0011 lacP pSB1A2 55
#21 C0012 LacI pSB1A2 1128
#22 I712074 pT7 pSB1AK8 46
#23 K145001 T7 RNA Pol. pSB1A2 2655
#24 J22106 recAp pSB1A2 192
#27 C0083 AspA pSB2K3 1518
#28 K112808 T4 phage lysis device (no promoter) pSB1A2 1785
#29 - CheZ pSB1AK3 728
#30 K117000 Lysis gene pSB1A2 144
#31 - LexA pSB1AK3 750
#33 - sulAp pSB1AK3 67
#34 - uvrAp pSB1AK3 96
#35 - recNp
#36 R0051 cI-repressed promoter pSB1A2 49
#37 C0051 cI repressor (LVA tagged) pSB1A2 \
#38 - RecA

Lab Diary

For convenience sake, each part (genes, promotors, etc) is represented by consecutive numbers (e.g. #20 for lac promotor). By pointing the mouse on the part number in the construct, you can find out the details of the part.


  • May
  • June
  • July
  • August
  • September
  • October

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'11/5/18 (Wed)

  • Making LB medium, 50×TAE, Tris-HCl (pH8.0)

'11/5/24 (Tue)

  • Making SOB medium, 0.5M EDTA (pH8.0)

'11/5/25(Wed)

  • Making TB(pH=6.7), LB plate

'11/5/26(Thu)

  • Making Mgaq(for SOB medium), Competent cell
  • TB filtration

'11/5/31(Tue)

  • iGEM parts resuspension + frozen stock (at -20℃)
  • Transformation
  • Overnight culture on LB plate (with 100ug/ml ampicilline)

'11/06/01(Wed)

  • Picking up colony and transfer to LB medium
  • Making LB medium

'11/06/02(Thu)

  • Miniprep
  • Making Glycerol(50%)(for cryopreservation)

'11/06/07(Tue)

  • Nanodrop
  • Transformation
  • Culture from frozen stock

'11/06/08(Wed)

  • Picking up colony

'11/06/09(Thu)

  • Miniprep

'11/06/13(Mon)

  • Planting Negative control
  • Making frozen stock
  • Transformation(BBa_E0240, #3, B0015, #5, #10, ,J31000, ,J44000)

'11/06/14(Tue)

  • Picking up colony
  • Making Mg reagent

'11/06/15(Wed)

  • Miniprep

'11/06/17(Fri)

  • Picking up colony(#3, B0015, B0040,#9, J31000, J44000)
  • Miniprep(#5)
  • Dissolution(#1, #2, K16500, #24)

'11/06/21(Tue)

  • Making frozen stock(#3, B0015, B0040, #9, J31000, J44000)
  • Passafe culture(#9, J31000)
  • Nanodrop again

'11/06/22(Wed)

  • Miniprep(#9, J31000)
  • Making competent cell

'11/06/24(Fri)

  • Digest (product of Miniprep 06/09 EScut)
  • Making agarose gel
  • Electrophoresis

'11/06/28(Tue)

  • Digest(product of Miniprep 06/09 EPcut)
  • Electrophoresis
  • Defrost and transformation(BBa_E0030, E0020, #14, I712052,#17）
  • Making 1×TAE, agarose gel, LB medium, LB plate(amp),

'11/06/29(Wed)

  • Picking up colony
  • Making LB broth

'11/07/01(Fri)

  • Digest(EPcut)
  • Miniprep(BBa_E0030, E0020, #14, I712052, #17)

'11/07/05(Tue)

  • Electrophoresis(product of digest 07/01)
  • Digest(#9, #14, #17, #3, #5)
  • Making antibiotic stock(1000×)(Km,Cm)

'11/07/06(Wed)

  • Gel extraction (pre)(BBa_I712052)
  • Picking up colony(Ba_J23119, #2, #24, #10)
  • Making agarose gel

'11/07/07(Thu)

  • Digest（BBa_E0240, #14, #17, #3, #5）
  • Defrost（BBa_E1010, K325101, #20, #21, #22, #23）
  • Transformation（BBa_K145001, #20, #21, #22）
  • Making LB plate（Cm,Km）

'11/07/08(Fri)

  • Miniprep(BBa_J23119, #2, #10, #24)

'11/07/11(Mon)

  • Gel extraction(#9 #14, #17)
  • Transformation(#1, #11, #20, #21, K325101, #22, #23)

'11/07/12(Tue)

  • Picking up colony

'11/07/13(Wed)

  • Frozen stock(#1(18A), #20(6G), #21(2O), #14(6N), #23(2F))
  • Picking up colony(#11)
  • Gel extraction(B#3, #5)
  • Digest (#2 SPcut)

'11/07/14(Thu)

  • Miniprep(#1, #11, #20, #21, #22, #23)
  • Electrophoresis(#2)
  • Gel extraction(#2, #3, #5)
  • Passafe(#11)
  • Making LB+amp

'11/07/15(Fri)

  • Ligation(#2-#9, #2-#3, #14-#5, #17-#5)
  • Transformation(#2-#9, #2-#3, #14-#5, #17-#5)
  • Dispensing Ligation Buffer
  • Frozen stock(#11)

'11/07/19(Tue)

  • Digest
  • Gel extraction
  • Making competent cell

'11/07/20(Wed)

  • Picking up colony
  • Making Master plate
  • Testing product of Miniprep
  • Transformation (#2, #27, #28)

'11/07/21(Thu)

  • Miniprep(#2-3, #2-9, #14-5, #17-5, #11, #20)
  • Testing product
  • Digest
    • SPcut : #20, #2-3
    • XPcut : #10, #11, #14-5, #17-5
  • Making TB

'11/07/22(Fri)

  • Frozen stock(#2, #2-3, #2-9, #14-5, #17-5）
  • Electrophoresis(#20_SP, #2-3_SP, #10_XP, #11_XP, #14-5_XP, #17-5_XP）
  • Digest
    • SPcut : #20
    • XPcut : #10, #11, #14-5, #17-5
  • Transformation(#27, #28, product of Miniprep 07/20)

'11/07/25(Mon)

  • Colony check(#2, #2-9)
  • Gel Extraction(#20_SP, #10_XP, #11_XP, #14-5_XP, #17-5_XP)
  • Ligation and Transformation(#20-#9, #3-#11, #3-#14-5, #3-#17-5)
  • Defrost Primer(200×, 10×)
  • Dispensing PCR Mix

'11/07/26(Tue)

  • Picking up colony and making master plate(#20-9, #3-11, #3-14-5, #3-17-5)
  • Colony PCR(#20-9, #3-11, #3-14-5, #3-17-5)
  • Digest(#14-5 XPcut,Ecut, #22 SPcut, #23 XPcut)
  • Ligation(#3-#14-5(Re), #3(Negative control))
  • Transformation(#15, #27, #28, #30, #3-14-5(Re), #3(Negative control))
  • Making reagent for TB(KOH, CaCl2, KCl, MnCl2)

'11/07/27(Wed)

  • Miniprep(#1, #2, #3, #10, #22, #24, #20-9, #3-11, #3-17-5)
  • Electrophoresis

'11/07/28(Thu)

  • Gel extraction(#14-5_XP, #22_SP, #23_XP)
  • Digest
    • SPcut : #1,#2, #3, #24
    • EScut : #3-11
    • XPcut : #3-17-5
  • Ligation(#3-#14-5)
  • Making TB bugger, LB plate, 0.3% LB plate

'11/07/29(Fri)

  • Cloning(lexA, cheZ)
  • Colony PCR(#3-#14-5)
  • Gel extraction(#1_SP, #2_SP, #3_SP, #24_SP, #3-11_ES, #3-17-5_XP, lexA, cheZ PCR product）
  • Miniprep(#30)
  • Digest(lexA, cheZ PCR product XP cut)

'11/08/01(Mon)

  • Nanodrop(#3-11(1), #1, #3, #24, #2, #3-17-5(4)M#30)
  • Digest(#3 SPcut, #5 EXcut, #9 XPcut, #30 XP cut)
  • Cloning(lexA, cheZ)
  • Gel extraction(#3, #9, #30, pSB1A2)
  • Ligation(#2-#3-17-5, #3-11-#5, #14-#5, #3-#23, #22-#9, #3-#30)
  • Transformation(#2-#3-17-5, #3-11-#5, #14-#5, #3-#23, #22-#9, #3-#30, #27(IPTG), #28)

'11/08/02(Tue)

  • Colony PCR(#2-#3-17-5, #3-11-#5, #14-#5, #3-#23, #22-#9)
  • Cloning(PCR)(#27, #28)
  • Gel extraction
  • PCR_2nd(lexA, cheZ, #27, #28)
  • Digest (#30 again)
  • Making competent cell(cheZ-, Wild Type)
  • Miniprep

'11/08/04(Thu)

  • Electrophoresis(product of ligation #2-3-17-5(2), #3-11-5(1,2), #14-5(2), #22-9(1,2,3))
  • Frozen stock(#2-3-17-5(2), #3-11-5(1,2), #14-5(2), #22-9(1,2))
  • Making Gel
  • Digest(#23 XPcut)
  • IPTG induct(#20-9)

'11/08/05(Fri)

  • Digest(#14-5(2) XPcut, #3-11-5(1) XPcut)
  • Cloning(PCR)(#27, #28, lexA, cheZ)

'11/08/08(Mon)

  • Defrost Frozen stock, Miniprep, Nanodrop and Electrophoresis(#5, #20, #23, #30)
  • Making Gel
  • Digest
    • XPcut : #27, #28, cheZ, lexA, #3-11-5(1), #23,#30
    • EScut : #6
    • EXcut : #7
    • SPcut : #7,#20
  • Defrost Frozen stock(#5, #14-5(2), #3-11-5(1))

'11/08/09(Tue)

  • Miniprep, Electrophoresis and Digest(#5, #14-5(2), #3-11-5(1))
  • Gel extraction and Nanodrop(#6, #27, #8, cheZ, lexA③, #3-11-5(1), #23, #7_EX, #20, #7_SP)
  • Ligation(#27-psB1A2, #3-27, #20-28, #28-psB1A2, #3-29, #29-psB1A2, #31-psB1A2, #3-#23, #1-#3-11-5(1), #2-#3-11-5(1), #24-#3-11-5(1), #3-#30)
  • Making LB plate

'11/08/10(Wed)

  • Colony PCR(#27-pSB1A2, #3-27, #20-28, #28-psB1A2, #3-29, #29-psB1A2, #31-pSB1A2, #3-#23, #1-#3-11-5(1), #2-#3-11-5(1), #24-#3-11-5(1), #3-#30）)
  • Cloning(lexA, cheZ)
  • Digest
    • EScut : #6
    • EXcut : #7
    • SPcut : #1, #2, #3, #7, #20
    • XPcut : #23
  • Defrost Primer

'11/08/11(Thu)

  • Gel extraction(#30, #14-5(insert,vector), #5, #3-11-5, cheZ, lexA)
  • Cloning(sulAp, uvrAp)
  • Miniprep(#3, #20-28, #31, #24-3-11-5, #3-30)

'11/08/12(Fri)

  • Colony PCR(#3-14-5(2), #6-5, #3-23)
  • Cloning(nested-PCR)(uvrAp, sulAp)
  • Check Electrophoresis
    • 8/11 PCR product：#27, #28
    • Miniprep product：#3-14-5(2), #5
  • Digest（#5 EXcut, #14 EScut）
  • Gel extraction
    • 8/11 digest product : #5, #14-5, #3-30, cheZ, lexA
    • 8/11 PCR product : #27, #28
    • 8/12 nested-PCR product : sulAp, uvrAp
  • Colony PCR (re) (#13-14-5)
  • Miniprep(ligation product : #6-5, #3-23)
  • Cloning(hotstart-PCR: uvrBp , cheZ)
  • Digest
    • XPcut:#27, #28, #6-5
    • EScut: sulAp, uvrAp, #3-23

'11/08/15(Mon)

  • Gel extraction(from previous digest products(8/12) : #27, #28, #33, #34, #4, #3-23)
  • Digest
    • XPcut: cheZ, uvrBp(PCR products)
    • EScut: #3-11-5 (to obtain pSB1AK3 EScut)
  • Assay of Cell lysis construction(pLuc-Lysis device) inducted by IPTG
  • Making Lysis buffer, Agarose gel
  • Gel extraction(from digest products XPcut: cheZ, uvrBp(PCR products) EScut: #3-11-5 to obtain pSB1AK3 EScut)
  • Ligation(#3-#14-5, #24-#3-17-5, #24-#3-11-5, #20-#28, #3-#27, #3-#29, #3-23-#5, #33-pSB1AK3, #34--pSB1AK3, #35--pSB1AK3)
  • Transformation(ligation products:WT, #2-9:cheZ-, #14, #17) -> 37 incubation

'11/08/16(Tue)

  • Colony PCR(#3-#14-5, #24-#3-17-5, #20-#28, #3-#27, #3-#29, #3-23-#5)
  • Culture (#20-#28, #5, #14, #17, #2-9, #2-4, #2-3-17-5, #1, #2)
  • Incubation (#24-#3-11-5, #33-pSB1AK3, #34-pSB1AK3, #35-pSB1AK3)
  • Colony PCR(additional)(previously incubated ligation products: 3, 8, 10)
  • hotstart PCR(sulAp, uvrAp, uvrBp)
  • Digest(SPcut: #3, Ecut:#3(control)) go to 37 incubation
  • Ligation retry(#3-#14-5, #3-#27, #3-#29, Negative control :#3)
  • Frozen stock(#14, #17)
  • Culture test(#14 on 0.3%Agar.(km))
  • Ethanol precipitation: 29.9ng/ul * 1000ul
  • Miniprep(Ligation products : #2-3-17-5, #14, #17, #1, #2)
  • Electrophoresis(Colony PCR products: 3, 8, 10)
  • Waking from frozen stock(#3, CheZ-, WT)

'11/08/17(Wed)

  • Hotstart-PCR (Change conditions a little)
    • From Genome
    • From nested-region
    • From parts
  • Making Competent Cell, SOB medium
  • Ethanol precipitation(WT: 40ng/ul)
  • Culture from master-plate(#6-5, #20-28, #3-27, #33, #24-3-11-5 #24-3-17-5) -> Miniprep
  • Assay(IPTG induced cell lysis device
  • Gel extraction(SPcut:#3)
  • Culture from Frozen stock(#1, #2, #2-3-17-5)
  • Electrophresis check(Loading only DNA size markers)
  • Miniprep(#3, #6-5, #20-28, #3-27, #33, #24-3-11-5 #24-3-17-5)

'11/08/18(Thu)

  • Digest(8/17#3, #33 SPcut, #14-5 XPcut, #33 Ecut, #3-27,#3-30,#20-28 EScut, pSB1K3 EPcut)
  • Hotstart-PCR (Change conditions a little)
    • From Genome : uvrBp, cheZ
    • From nested-region : uvrA
  • Culture from 0809 master-plate ⑪ (#2-3-11-5) → Miniprep.
  • Making LB plate & tube with various agarose conc. (plate: 0.5, 0.7, 0.9%×2 each, tube: 0.5,1.0,1.5,2.0,3.0%×2 each)(selection: Kanamycin)
  • Gel extraction(8/17#3_SP,#33_SP, #14-5_XP, #33_E, #3-27_ES, pSB1K3 _EP)
  • Miniprep(#1, #2, #2-3-11-5, #2-3-17-5)
  • Digest(#1,#2 SPcut, #2-3-17-5 XPcut, #2-3-11-5 SPcut(check), EPcut(selection), #3-30, #20-28 EScut, 0817 Gel extracted PCR product (uvrBp) EScut, 0818PCR products (uvrAp, uvrBp, cheZ) ES&XPcut)
  • Ligation (8/17#3-#14-5, 8/17#3-8/15#29, #33-#3-11-5, 8/15#34-pSB1AK3, #3-27-#5, #20-28-pSB1AK3, #28-pSB1AK3)
  • Culture for frozen stock in eppendolf tube

'11/08/19(Fri)

  • ColonyPCR(from ligation products)(#3-14-5, #3-29, #33-3-11-5, #34-pSB1AK3, #3-27-5)
  • Gel extraction(08/18's digest products:#1_SP, #2_SP, #2-3-11-5_SP, #2-3-17-5_XP, cheZ_XP, #2-3-11-5_EP, 8/18uvrBp_ES, 8/17uvrBp_ES, uvrAp_ES)
  • Colony PCR retry(#3-29, #34-pSB1AK3)
  • Frozen Stock
  • Check the list of frozen stock
  • Assay of recAp from biobrick parts using RFP construct & transilluminator
  • Electrophoresis(#3, #3-27, #3-30, #6-5, #7, #20-28, #24-3-11-5, #24-3-17-5, #33)

'11/08/22(Mon)

  • Making SOB medium
  • Culture for Miniprep
    • From frozen stock(#1, #2, #7, #2-3-17-5(2), #3-30)
    • From master plate(#3-14-5, #33-3-11-5, #34-pSB1AK3, #3-27-5)
    • From master plate retry( at 13:50 OD600=0)
    • From ligation products (for mistake above)
  • Miniprep, Nanodrop and Electrophoresis
    • From frozen stock(#1, #2, #2-3-17-5(2), #3-30) #7 was a little bit weak
    • From ligation products(#7, #3-14-5, #3-27-5, #34, #33-3-11-5)
  • Digest
    • From Frozen Stock (#7_EX, #30_XP, #2-3-17-5_XP, #3-30_ES)
    • From Master plate(#3-14-5_XP, #34-pSB1AK3_SP, #3-27-5_XP)
  • Sulvage #33 using protocol of transformation
  • Culture for Frozen stock
    • From Master plate(#24-3-17-5, #14-5), 700ul, in eppendolf tube

'11/08/23(Tue)

  • Gel extraction
    • from O.N. digest products(#7_EX, #30_XP, #2-3-17-5_XP, #3-30_ES, #3-14-5_XP, #34-pSB1AK3_SP, #3-27-5_XP)
  • Frozen stock
    • From O.N. culture in eppendolf tube 700ul(#24-3-17-5, #14-5)
    • From O.N. culture(for Miniprep)(#34, #3-27-5, #33-3-11-5)
  • Miniprep
    • retry from master plate #3-14-5(278.1 ng/ul), #33-3-11-5(188.9ng/ul), #34-pSB1AK3(89.6ng/ul), #3-27-5(327.0 ng/ul) O.N. culture
  • Digest (#14-5 XPcut, #3-27-5 Xcut, Pcut (check), #3-27-5 EScut (sulvage) )
  • Ligation (#14-#5, #3-30-#5, #3-#30, #34-#3-11-5 #20-#28)
  • #33 sulvage (isolation culture)
  • Making culture(LB amp, LB)

'11/08/24(Wed)

  • Gel extraction & Electrophoresis(#3-27-5_ES, #14-5_XP, #3-27-5_X, *PCR for amplification (or cloning) -> PCR clean-up system -> digest
    • #27 (82.1 ng/ul, total = 30 ul) -> XP cut
    • #28 (104.4 ng/ul, total = 30 ul) -> XP cut
  • Making plate and amp stock　->4℃
    • amp 32
    • km 11 (km conc. = 3/4 * that it was)
  • Miniprep and Nanodrop
    • #33 32.1 ng/ul <- there is probability that culture volume was not enough ->transfromation
  • Luciferase assay reagents prep
    • D-Luciferin Solution : 1ml*10 + 100ul*10 at -80℃
    • Coelenterazine Solution : 100ul * 10 at -20℃
    • Lysis buffer : 500ul * 10 at -20℃
  • Ligation(#3-30-#5, #3-11-5-#34, #30-#3, #28-#20, #35-pSB1AK3, #2-3-11-5-pSB1K3, #14-5-#3 and Negative control)

'11/08/25(Thu)

  • Renilla luciferase assay (with #2-3-17-5 culture)
  • Gel extraction (#27, #28 XPcut)
  • Ligation (0824 retry & #20-#28, #3-#27)
  • Transformation (Ligation products into WT competent cells & #14-5 miniprep product into WT cell (line-drawing spread), #33 miniprep product into ΔcheZ cell (line-drawing spread))
  • Digest (#14 XPcut)
  • Passage(#2-3-17-5, WT, ΔcheZ, WT in eppen, ΔcheZ in eppen)

'11/08/26(Fri)

  • Renilla luciferase assay (with #2-3-17-5 culture)
  • Colony PCR (#33 Miniprep transformations)
  • Gel extraction (#14_XP)
  • Ligation (retry) (using today's competent cells, 0802 ΔcheZ cells)
  • Cloning (cheZ)
  • Digest (#5 EXcut, cheZ PCR products XPcut)
  • Passage in a shaker(#2-3-17-5, WT, ΔcheZ)
  • Making competent cell, LB broth
  • Contamination check (0817 comp., 0802 comp., 0802 ⊿cheZ comp. spread on LB (amp) plates)

'11/08/27(Sat)

  • Ligation & Transformation (0826 ①②③⑤⑧⑨) (into Nippon Gene comp.)
  • Contamination check (WT FS, ΔcheZ Frozen stock)
  • Waking from Frozen stock(WT, ΔcheZ)

'11/08/28(Sun)

  • Making ΔcheZ comp., LB amp plates, TB
  • Colony PCR & master plate preparation -> Failure!!
  • Gel extraction (#29_XP, #5_EX)
  • Miniprep and frozen stocks (#33, #14-5)
  • Digest (#20 SPcut, #14-5 XPcut)
  • Waking from frozen stock (WT, ⊿cheZ, #20, #14-5)

'11/08/29(Mon)

  • PCR purification -> digest with XP(#27, #28)
  • Miniprep(#20, #14-5)
  • Gel extraction(#14-5, #20)
  • Ligation (higher I/V ratio, into cheZ comp.)(#3-#14-5(retry), #2-3-11-5 to pSB1K3 (retry), #35-pSB1AK3, (#33,#34)-#3-17-5)

'11/08/30(Tue)

  • Making LB amp, Aspartic acid solution (10mM), H2O2 solution (10mM)
  • Gel extraction -> PCR was failed!(#27,#28)
  • There was trash (from PCR) below
  • Colony PCR(#3-#14-5, #2-3-11-5 to pSB1K3, #35-pSB1AK3, (#33,#34)-#3-17-5)
  • PCR -> check (5uL EP -> PCR clean-up(#27,#28)
  • Digest(#20_SP, pSB1AK3_EP, #3_SP)
  • EP for MP check(#14-5, #20)
  • Ligation(#3-#29, pSB1AK3-#2-3-11-5 on Amp/Km plate, #3 (Negative control), pSB1AK3 (Negative control) on Amp/Km plate
  • Transformation(#2-9 into cheZ-) -> Line-drawing plating
  • SOS stress (UV irradiation or H2O2)(#24-3-11-5, #33-3-11-5)
  • culture for Miniprep from master plate(#34-3-17-5)
  • Waking from frozen stock(#24-3-11-5, #33-3-11-5)

'11/08/31(Wed)

  • Making LB plates(amp, km)
  • Luciferin reagent preparation
  • Flask A.C. for making competent cells
  • Miniprep (#34-3-17-5)
  • Gel extraction(#20_SP, pSB1AK3_EP, #3_SP)
  • PCR -> clean-up -> XPcut(#27, #28, #14-5, #3-30)
  • Waking from frozen stock(#2-9 (WT), #24-3-17-5)
  • Plating from liquid culture(#2-3-17-5)
  • Ligation(#20-#3-17-5, #2-3-11-5-pSB1AK3, #3-#14, #20-#28, #3-#27, #3-#29, #35-pSB1AK3, #3-#14-5, #3-30-#5, #33-#3-17-5 & Negative control)
  • Transformation
    • Plasmid isolation(#34-3-17-5(MP;1:9gradient))
    • Part cloning(#36, #37)
    • Contamination check(pSB1AK3(EPcut), pSB1K3(EPcut))
    • Titer check(#3-17-5(Takara comp.,1:9gradient), #2-3-11-5(ΔcheZ comp.0802, 1:9gradient), #2-3-11-5(ΔcheZ comp.0830, 1:9gradient))
    • Ligation products

'11/09/01(Thu)

  • Colony PCR
    • #34-3-17-5(MP), #3-14, #3-29, #35-pSB1AK3, #3-14-5, #37(from Part cloning)
  • Gel extraction
    • #27_XP ,#28_XP(0830); #27_XP, #28_XP, #3-30_XP -> failed!, #14-5_XP(0831)
  • Making medium(SOB, LB amp plate), competent cells (WT & ΔcheZ)
  • Miniprep(#3, #20)
  • PCR and Column purification
    • #3-14, #3-29, #3-14-5 (Recovery from colony PCR tubes!)
  • Digest
    • #3 -> Failed (Mixed with DMSO!), #20
    • #3-14, #3-29, #3-14-5
  • Ligation
    • #3-27, #20-#3-17-5
  • Transformation
    • #36 into Nippon Gene comp.
    • Ligation products into Takara comp. (including N.C.)
    • Titer check(MP product #3 into 0901 WT comp., MP product #20 into 0901 ΔcheZ comp.)
  • Mobility assay(#14-5 (km resistance) WT/ΔcheZ cells)

'11/09/02(Fri)

  • Making medium and reagent(LB 120ml(0.125, 0.25, 0.5% Agar, M9 stock(20×), M9 200ml, Vitamins Solution (100×))
  • Gel extraction -> Digest products were OK but extraction was failed.
    • #20_SP, #3-14_XP, #3-14-5_XP, #3-29_XP
  • Colony PCR & Master plate(#20-3-17-5, #3-27)
  • Miniprep(#3, #20, #37, #3-14, #3-29, #3-14-5, #20-3-17-5, #3-27, ΔcheZ)
  • Hot-start PCR & Column purification -> PCR products were OK but all purified products were lost.
    • #20-3-17-5, #3-27 (Recovery from colony PCR tubes)
    • #3-14, #3-14-5, #3-29 (from miniprep products)
  • PCR & Column purification
    • #20-3-17-5, #3-27, #3-14-5, #3-29 (from miniprep products)
  • Digest
    • #3_SP, #20_SP, #37_ES, #3-14_ES, #3-29_ES, #3-14-5_XP, ΔcheZ_E/P (from MP products)
    • #20-3-17-5_XP, #3-27_ES, #3-14-5_XP, #3-29_ES (from PCR products)

'11/09/03(Sat)

  • Gel extraction
  • Ligation & Transformation
    • #20-#3-14-5, #2-#3-14-5, #3-27-#5 into Nippon Gene comp. (+ High Competent Broth 240ul)
    • #3-27-#5, #37-#5 into 0902 WT comp.（+ Takara SOC broth 500ul）
  • Assays
    • Mobility assay (.5%, .25%, .125%)
    • Asp assay
  • Part Cloning (#36)

'11/09/04(Sun)

  • Hot-start PCR Pre-Mix (4×) preparation (Buffer + dNTPs + THUNDER Taq)
  • Colony PCR(#20-3-14-5, #3-27-5, #3-29-5, #37-5)
  • Colony Diffusion Assays
  • GFP Live-Imaging Assays
  • Ligation & Transformation (retry)
    • #2-#3-14-5, #3-27-#5, #3-29-#5, #37-#5, #20-#28 into 0901 WT comp.
    • #36 into Takara comp.
    • #2, #5 as Negative Controls into 0901 WT comp.
  • Culture for Miniprep(#20-3-14-5, #20)
  • Digest(#20)

'11/09/05(Mon)

  • Colony Diffusion Assays (#2-9) WT,cheZ-
  • Gel extraction(#20)
  • Luciferase Assays
    • Calibrations (Firefly Luciferase standards)
    • Background Assay
    • Dual Luciferase Assay(#20-3-14-5, #20-3-17-5)
  • Colony PCR(#2-#3-14-5, #3-27-#5, #3-29-#5, #37-#5, #20-#28, #36)
  • PCR -> clean-up -> Digest(#3-29-#5, #37-5)
  • IPTG-inducted Cell lysis test(IPTG+, LB plate (Positive control))
  • Miniprep(#20-3-14-5, #37-5, #36)
  • Making M9 -> Asp -> all failed(.125%, .25% Agar)
  • Culture for Miniprep(#2-3-14-5)

'11/09/06(Tue)

  • Gel extraction(#37-5, #3-29-5)
  • Miniprep(#2-3-14-5,　#20-3-14-5, #20-3-17-5, #36, #3-29-5, #37-5)
  • PCR (retry) (Hot-start & Normal PCR) -> Purification -> Digest(#3-29-5_XP, #37-5_ES)
  • PCR (cloning retry)(lexA, uvrBp)
  • Ligation & Transformation
    • #3-27-#5, #3-30-#5, #2-3-11-5-pSB1AK3
    • #5, pSB1AK3 (N.C.)
  • Culture for GFP live-imaging (#2-9 WT/cheZ-) -> 4℃ storage
  • Ninhydrin sol. preparation (Not dissolvable!) -> 4℃ storage

'11/09/07(Wed)

  • Colony PCR(#3-27-5, #3-30-5)
  • Digest(#36_SP)
  • Culture from master plate(#36, #3-29-5, #37-5)
  • Miniprep(#36, #3-29-5, #37-5, #3-27-5 (failed))
  • PCR purification & Digest(lexA_XP, uvrBp_ES)
  • Gel extraction(#3-29-5_XP, #37-5_ES, pSB1AK3_EP, #36_SP)
  • PCR amplification -> Digest(#3-14-5, #3-29-5, #37-5, #3-27-5)
  • Digest(#36_SP (re), #2_SP, #2-3-17-5_EX, #3-14-5_ES, #34_SP)
  • Culture for Miniprep(#2, #34, #36, #3-27-5, #3-14-5, #2-3-17-5)

Ligation & Transformation

    • #33-#3-14-5, #34-#3-14-5, #36-#3-14-5, #2-#3-29-5, #20-#3-29-5, #3-#37-5, #2-3-11-5-pSB1AK3, #1-#3-17-5
    • #33, #34, #36, #1, #2, #20, #3, pSB1AK3 (N.C.)
  • Ninhydrin stock solution preparation
    • Asp conc. check (it took 5 min at 80C)
  • Making M9 medium(.25, .125% Agar.)

'11/09/08(Thu)

  • Making competent cell (WT), LB(amp) plate
  • Frozen stocks(#36, #3-14-5, #3-27-5)
  • Colony PCR(#33-3-14-5, #34-3-14-5, #36-3-14-5, #20-3-29-5, #2-3-11-5(psB1AK3))
  • Miniprep(#2, #34, #36, #2-3-17-5, #3-27-5, #3-14-5)
  • Gel extraction
    • PCR product: #35_SP, #31_XP, #3-14-5_XP, #3-27-5_XP, #3-29-5_XP, #37-5_XP
    • Miniprep product: #2_SP, #34_SP, #36_SP, #2-3-17-5_EX, #3-14-5_ES
  • PCR amplification(#34-3-14-5, #36-3-14-5, #20-3-29-5, #20-3-14-5)
  • PCR for Sequencing -> Store at -20C(#33-3-11-5, #34-3-14-5, #20-3-29-5, #37-5)
  • Ligation(#35_ES-psB1AK3_ES, #31_XP-psB1A2_XP, #31_XP-psB1AK3_XP, #3-#37-5, #36-3-29-5, #3-14-5_ES-#2-3-17-5_EX, #20-#3-27-5)
  • Culture for Miniprep(#34-3-17-5, #36-3-14-5, #20-3-29-5, #2-3-11-5)
  • Digest
    • PCR products: #34-3-14-5_ES, #36-3-14-5_ES, #20-3-14-5_ES
    • pSB1C3_EP
  • Asp Gradient test

'11/09/09(Fri)

  • Making competent cell (WT), TB
  • Colony PCR(#35-pSB1AK3 (failed), #31-pSB1AK3, #3-#37-5, #36-#3-29-5, #3-14-5-#2-3-17-5, #20-3-27-5(failed))
  • PCR amplification
    • From Miniprep : #3-14-5, #3-17-5
    • From Colony PCR : #31, #3-37-5, #36-3-29-5, #3-14-5-2-3-17-5 (failed)
  • Digest
    • Miniprep product: #3-11-5_EP
    • PCR-purified product: #3-14-5_EP, #3-17-5_EP, #31_EP, #3-37-5_XP, #36-3-29-5_ES
  • Miniprep(#34-3-14-5, #36-3-14-5, #20-3-29-5, #2-3-11-5 (pSB1AK3))
  • Gel extraction(#34-3-14-5_ES, #36-3-14-5_ES, #20-3-14-5_ES, #3-11-5_EP)
  • Ligation
    • #3-11-5_EP-pSB1C3_EP, #29_XP-pSB1AK3_XP
    • #36-3-14-5_ES-#2-3-17-5_EX, #20_SP-#3-27-5_XP(retry)
  • Comp. check
    • #2-3-11-5 (pSB1AK3) into 0908 WT comp.
    • #20-3-29-5 into 0909 WT comp.
    • #20-3-29-5 into 0802 cheZ- comp.
  • Part cloning(BBa_J04450 (IPTG-induced RFP reporter on pSB1C3))
  • Culture for Miniprep(#31-pSB1AK3, #3-37-5, #36-3-29-5, #3-14-5-2-3-17-5)
  • UV irradiation -> Luciferase Assay(#24-3-17-5, #34-3-17-5)
  • Asp Gradient test
  • CleanSEQ -> Sequencing(#33-3-11-5, #34-3-14-5, #20-3-29-5, #37-5)

'11/09/10(Sat)

  • Culture plate storage (4℃)
  • Miniprep(#31-pSB1AK3, #3-37-5, #36-3-29-5, #3-14-5-2-3-17-5)
  • Gel extraction(#3-14-5_EP, #3-17-5_EP, #31_EP, #3-37-5_XP, #36-3-29-5_ES)
  • Colony PCR(#36-3-14-5-#2-3-17-5 (Maybe Ligation was FAILURE , estimated length is 1000-1500 by electrophoresis))
  • Ligation(#20_SP-#3-37-5_XP)

'11/09/11(Sun)

  • Digest from Miniprep products
    • #3-14-5-2-3-17-5_XP
    • #36-3-14-5_SP, #20_SP -> O.N.
  • Colony PCR
    • #3-11-5 (pSB1C3), #29 (pSB1AK3), BBa_J04450 (pSB1C3)
    • #20-#3-27-5(failed), #20-#3-37-5, #36-#3-14-5-2-3-17-5
  • Gel extraction(#3-14-5-2-3-17-5_XP)
  • Culture for Miniprep
    • From Master plate : #20-3-37-5, #29 (pSB1AK3), BBa_J04450 (pSB1C3)
    • From plate stock : #20
    • From frozen stock : #2-3-17-5
  • Miniprep( #36-3-14-5-2-3-17-5)
  • Ligation(#20_SP-#3-14-5-2-3-17-5_XP, #1_SP-#3-14-5-2-3-17-5_XP)
  • Direct Ligation
    • i.PCR amplification -> Digest for >1h -> Heat kill for 20min (#20-3-37-5_XP, #36-3-14-5-2-3-17-5_ES)
    • ii.Digest for >1h -> Heat kill for 20min (#36-3-14-5-2-3-17-5_SP, pSB1A3_EP, #2-3-17-5_EX)
    • iii.Ligation for 30min (#20-3-14-5_ES-#2-3-17-5_EX, #36-3-14-5-2-3-17-5_SP-#20-3-37-5_XP, #36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1A3_EP)
    • iv.Transformation
  • Making 0.125% agar LB amp plate (IPTG 1, 10, 30, 100uM)
  • Cell Diffusion Assay
    • #20-3-29-5 WT (IPTG 1, 10, 30, 100uM)
    • #20-3-29-5 cheZ-/- (IPTG 1, 10, 30, 100uM)
    • WT
    • cheZ-/-

'11/9/12 (Mon)

  • Colony PCR
    • #20-#3-14-5-2-3-17-5
    • #20-3-14-5-#2-3-17-5
    • #1-#3-14-5-2-3-17-5
    • #36-3-14-5-2-3-17-5-#20-3-37-5 (no colony)
    • #36-3-14-5-2-3-17-5-#20-3-37-5-pSB1A3 (PCR failed?)

Note: #20-3-37-5 colonies on the master-plate are RED. Maybe #2-3-11-5...
Note: #36-3-14-5-2-3-17-5-#20-3-37-5-pSB1A3 PCR amplification doesn't work. Seems failed extension...

  • PCR amplification
    • From master plate : #20-3-37-5
    • From 09/11 Miniprep product : #36-3-14-5-2-3-17-5

Note: Only one #20-3-37-5 colony is NOT RED.

  • Miniprep(#20, #29 (pSB1AK3), #2-3-17-5, #2-3-37-5, BBa_J04450 (pSB1C3))
  • Digest
    • From PCR : #36-3-14-5-2-3-17-5_ES, #20-3-37-5_XP
    • pSB1A3_EP, pSB1C3_EP
    • From Miniprep : #20-3-37-5_XP
    • J04450_EP, *#2-9_XP, #20-3-29-5_ES
  • Gel extraction
    • #20_SP, #36-3-14-5_SP
    • From OverNight digests : #2-3-17-5_EX
    • #36-3-14-5-2-3-17-5_ES
    • From today's PCR digests : #20-3-37-5_XP
  • Culture for Miniprep(#20-3-37-5 from 0911 master plate)
  • Dual Luciferase Assay(#20-3-14-5-2-3-17-5, #1-3-14-5-2-3-17-5, #36-3-14-5-2-3-17-5-20-3-37-5-pSB1A3)
  • Ligation
    • #36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1A3_EP
    • #36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1C3_EP
    • #3-27-5_XP-#20_SP
    • #36-3-29-5_ES-#20-3-37-5_XP-pSB1AK3_EP

'11/9/13 (Tue)

  • Colony PCR
    • #36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1A3_EP
    • #36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1C3_EP (failed)
    • #3-27-5_XP-#20_SP (failed)
    • #36-3-29-5_ES-#20-3-37-5_XP-pSB1AK3_EP
  • Culture for Luciferase Assay
    • #36-3-14-5-2-3-17-5-20-3-37-5(pSB1A3, pSB1C3) (IPTG 0, 1, 10, 38, 100uM)
    • #20-3-14-5-2-3-17-5 (IPTG 0, 1, 10, 38, 100uM)
    • #1-3-14-5-2-3-17-5 (IPTG 0uM)

Note: Seemes OD600 should NOT exceed 0.4~0.5, or intracellular luciferase will be saturated.

  • Miniprep.
    • #20-3-14-5-2-3-17-5
    • #1-3-14-5-2-3-17-5
    • #20-3-37-5 (from a non-red colony on the master plate)
  • Gel extraction
    • #20-3-37-5_XP (from non-red colony)
    • #20-3-29-5_ES(failed)
    • #2-9_XP, pSB1C3_EP

Note: #20-3-29-5 was not cut!

  • Dual Luciferase Assay
    • #1-3-14-5-2-3-17-5 (IPTG 0uM)
    • #20-3-14-5-2-3-17-5 (IPTG 0, 1, 10, 38, 100uM)
    • #36-3-14-5-2-3-17-5-20-3-37-5 (#2-3-11-5?) (IPTG 0, 1, 10, 38, 100uM)
  • Sequencing preparation
    • #20-3-37-5 (from a non-red colony on the master plate)
    • #20-3-29-5
    • #3-27-5
    • #31
  • Digest
    • #20_E
    • #3-29-5_S (from MP products) (cut check)
  • Ligation
    • #20_SP-#3-27-5_XP
  • Transformation
    • #3-14-5-2-3-17-5
    • #1-3-14-5-2-3-17-5
    • #20-3-14-5-2-3-17-5 (for plate stock)
    • #20-3-37-5 (from a non-red colony on the master plate) (for plasmid check)

'11/09/14(Wed)

  • Colony PCR
    • #3-27-5_XP-#20_SP (failed)
  • Culture for Luciferase Assay (from plate stocks)
    • #20-3-14-5-2-3-17-5 (IPTG 0, 1, 10, 38, 100uM)
    • #1-3-14-5-2-3-17-5 (IPTG 0uM)
  • Digest
    • #3-14-5, #3-17-5, #3-14-5-2-3-17-5, #1-3-14-5-2-3-17-5, #20-3-14-5-2-3-17-5 (EPcut)
    • #20_SP
  • Culture for Miniprep.
    • From plate stock : #20, #20-3-14-5-2-3-17-5, #3-14-5-2-3-17-5, #3-17-5
    • From 09/11 master plate : #3-11-5 (pSB1C3)
  • Part cloning(#19: BBa_R0010)
  • Ligation
    • #20_SP-#3-29-5_XP
    • #36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1C3
  • Making WT competent cell
  • Dual Luciferase Assay
    • #1-3-14-5-2-3-17-5 (IPTG 0uM)
    • #20-3-14-5-2-3-17-5 (IPTG 0, 1, 10, 38, 100uM)
  • Asp TLC Assay
  • Asp Diffusion Assay (0.25% Agar)
  • Fumaric Acid (Fum) Sol. preparation
  • Sequencing
    • ①F⑤R#20-3-37-5 (from a non-red colony on the master plate)
    • ②F⑥R#20-3-29-5 (#2-3-11-5?)
    • ③F⑦R#3-27-5
    • ④F⑧R#31

Retrieved from "http://2011.igem.org/Team:UT-Tokyo/LabNote"