Team:UT-Tokyo/LabNote

From 2011.igem.org

(Difference between revisions)
Line 245: Line 245:
*Electrophoresis
*Electrophoresis
*Defrost and transformation(BBa_E0030,E0020,I712019,I712052,J52008）
*Defrost and transformation(BBa_E0030,E0020,I712019,I712052,J52008）
-
*Making 1×TAE, agalose gel, LB medium, LB plate(amp),
+
*Making 1×TAE, agarose gel, LB medium, LB plate(amp),
=='11/06/29(Wed)==
=='11/06/29(Wed)==
Line 251: Line 251:
*Making LB broth
*Making LB broth
 +
=='11/07/01(Fri)==
 +
*Digest(EPcut)
 +
*Miniprep(BBa_E0030,E0020,I712019,I712052,J52008)
 +
=='11/07/05(Tue)==
 +
*Electrophoresis(product of digest 07/01)
 +
*Digest(E0240,I712019,J52008,B0032,B0014)
 +
*Making antibiotic stock(1000×)(Km,Cm)
 +
 +
=='11/07/06(Wed)==
 +
*Gel  extraction (pre)(BBa_I712052)
 +
*Picking up colony(Ba_J23119,J23118,J22106,E0040?)
 +
*Making agarose gel
 +
 +
=='11/07/07(Thu)==
 +
*Digest（BBa_E0240,I712019,J52008,B0032,B0014）
 +
*Defrost（BBa_E1010,K325101,K145001,I712074,R0011,C0012）
 +
*Transformation（BBa_K145001,I712074,R0011,C0012）
 +
*Making LB plate（Cm×10,Km×9）
 +
 +
=='11/07/08(Fri)==
 +
*Miniprep(BBa_J23119,J23118,J22106,E0040)
 +
 +
=='11/07/11(Mon)==
 +
*Gel extraction(E0240,I712019,J52008)
 +
*Transformation(J23119,R0011,C0012,E1010,K325101,K145001,I712074)
 +
 +
=='11/07/12(Tue)==
 +
*Picking up colony
 +
 +
=='11/07/13(Wed)==
 +
*Frozen stock(J23119(18A),R0011(6G),C0012(2O),I712079(6N),K145001(2F))
 +
*Picking up colony(E1010)
 +
*Gel extraction(B0032,B0014)
 +
*Digest (J23118 SPcut)
 +
 +
=='11/07/14(Thu)==
 +
*Miniprep(E1010,J23119,K145001,I712074,R0011,C0012)
 +
*Electrophoresis(J23118)
 +
*Gel extraction(J23118,B0032,B0014)
 +
*Passafe(E1010)
 +
*Making LB+amp
 +
 +
=='11/07/15(Fri)==
 +
*Ligation(J23118-E0240,J23118-B0032,I712019-B0014,J52008-B0014)
 +
*Transformation(J23118-E0240,J23118-B0032,I712019-B0014,J52008-B0014)
 +
*Dispensing Ligation Buffer
 +
*Frozen stock(E1010)
 +
 +
=='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 (<nowiki>#</nowiki>2,<nowiki>#</nowiki>27,<nowiki>#</nowiki>28)
 +
 +
=='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)
 +
*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）
 +
*Making TB
 +
 +
=='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）
 +
*Electrophoresis(<nowiki>#</nowiki>20,<nowiki>#</nowiki>2-3 SPcut　<nowiki>#</nowiki>10,<nowiki>#</nowiki>11,<nowiki>#</nowiki>14-5,<nowiki>#</nowiki>17-5 XPcut）
 +
*Digest(<nowiki>#</nowiki>20 SPcut, <nowiki>#</nowiki>10,<nowiki>#</nowiki>11,<nowiki>#</nowiki>14-5,<nowiki>#</nowiki>17-5 XPcut)
 +
*Transformation(<nowiki>#</nowiki>27, <nowiki>#</nowiki>28, product of Miniprep 07/20)
 +
 +
=='11/07/25(Mon)==
 +
*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)
 +
*Ligation and Transformation()
 +
*Defrost Primer(200×, 10×)
 +
*Dispensing PCR Mix
 +
 +
=='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)
 +
*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)
 +
*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))
 +
*Making reagent for TB(KOH,CaCl2,KCl,MnCl2)
 +
 +
=='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)
 +
*Electrophoresis
 +
 +
=='11/07/28(Thu)==
 +
*Gel extraction(<nowiki>#</nowiki>14-5 XPcut, <nowiki>#</nowiki>22 SPcut, <nowiki>#</nowiki>23 XPcut)
 +
*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)
 +
*Ligation(<nowiki>#</nowiki>3-<nowiki>#</nowiki>14-5)
 +
*Making TB bugger, LB plate, 0.3% LB plate
 +
 +
=='11/07/29(Fri)==
 +
*Cloning(lexA,cheZ)
 +
*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）
 +
*Miniprep(<nowiki>#</nowiki>30)
 +
*Digest(lexA, cheZ PCR product XP cut)
=='11/9/12 (Mon)==
=='11/9/12 (Mon)==

Revision as of 00:02, 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

Please enable Javascript to view this calendar.

'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,B0032,B0015,B0014,E0040,J31000,J44000)

'11/06/14(Tue)

  • Picking up colony
  • Making Mg reagent

'11/06/15(Wed)

  • Miniprep

'11/06/17(Fri)

  • Picking up colony(B0032,B0015,B0040,E0240,J31000,J44000)
  • Miniprep(B0014)
  • Dissolution(J23119,J23118,K16500,J22106)

'11/06/21(Tue)

  • Making frozen stock(B0032,B0015,B0040,E0240,J31000,J44000)
  • Passafe culture(E0240,J31000)
  • Nanodrop again

'11/06/22(Wed)

  • Miniprep(E0240,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,I712019,I712052,J52008）
  • 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,I712019,I712052,J52008)

'11/07/05(Tue)

  • Electrophoresis(product of digest 07/01)
  • Digest(E0240,I712019,J52008,B0032,B0014)
  • Making antibiotic stock(1000×)(Km,Cm)

'11/07/06(Wed)

  • Gel extraction (pre)(BBa_I712052)
  • Picking up colony(Ba_J23119,J23118,J22106,E0040?)
  • Making agarose gel

'11/07/07(Thu)

  • Digest（BBa_E0240,I712019,J52008,B0032,B0014）
  • Defrost（BBa_E1010,K325101,K145001,I712074,R0011,C0012）
  • Transformation（BBa_K145001,I712074,R0011,C0012）
  • Making LB plate（Cm×10,Km×9）

'11/07/08(Fri)

  • Miniprep(BBa_J23119,J23118,J22106,E0040)

'11/07/11(Mon)

  • Gel extraction(E0240,I712019,J52008)
  • Transformation(J23119,R0011,C0012,E1010,K325101,K145001,I712074)

'11/07/12(Tue)

  • Picking up colony

'11/07/13(Wed)

  • Frozen stock(J23119(18A),R0011(6G),C0012(2O),I712079(6N),K145001(2F))
  • Picking up colony(E1010)
  • Gel extraction(B0032,B0014)
  • Digest (J23118 SPcut)

'11/07/14(Thu)

  • Miniprep(E1010,J23119,K145001,I712074,R0011,C0012)
  • Electrophoresis(J23118)
  • Gel extraction(J23118,B0032,B0014)
  • Passafe(E1010)
  • Making LB+amp

'11/07/15(Fri)

  • Ligation(J23118-E0240,J23118-B0032,I712019-B0014,J52008-B0014)
  • Transformation(J23118-E0240,J23118-B0032,I712019-B0014,J52008-B0014)
  • Dispensing Ligation Buffer
  • Frozen stock(E1010)

'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(#20,#2-3 SPcut　#10,#11,#14-5,#17-5 XPcut）
  • Making TB

'11/07/22(Fri)

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

'11/07/25(Mon)

  • Colony check(#2,#2-9)
  • Gel Extraction(#20 SPcut #10,#11,#14-5,#17-5 XPcut)
  • Ligation and Transformation()
  • 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 XPcut, #22 SPcut, #23 XPcut)
  • Digest(#1,#2,#3,#24 SPcut, #3-11 EScut, #3-17-5 XPcut)
  • 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 and Agarase処理　(#1,#2,#3,#24 SPcut, #3-11 EScut, #3-17-5 XPcut, lexA,cheZ PCR product）
  • Miniprep(#30)
  • Digest(lexA, cheZ PCR product XP cut)

'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
    • #20-3-37-5 from Master plate
    • #36-3-14-5-2-3-17-5 from 0911 MP product

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
    • #36-3-14-5-2-3-17-5_ES (from PCR)
    • #20-3-37-5_XP (from PCR)
    • pSB1A3_EP
    • pSB1C3_EP
    • #20-3-37-5_XP (from MP) -> O/N
    • J04450_EP -> O/N
    • #2-9_XP -> O/N
    • #20-3-29-5_ES -> O/N
  • Gel extraction
    • #20_SP
    • #36-3-14-5_SP
    • #2-3-17-5_EX (from O/N digests)
    • #36-3-14-5-2-3-17-5_ES
    • #20-3-37-5_XP (from today's PCR digests)
  • Culture for Miniprep.
    • #20-3-37-5 from 0911 master plate -> O/N
  • 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 -> O/N
    • #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 ext.
    • #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 -> O/N
    • #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)

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