Team:UT-Tokyo/LabNote

From 2011.igem.org

(Difference between revisions)
Line 12: Line 12:
! Number !! Reg.ID !! Content !! Plasmid !! Length (bp)
! Number !! Reg.ID !! Content !! Plasmid !! Length (bp)
|-  
|-  
-
| #1 || J23119 || c.Promoter (Strong) || pSB1A2 || class="number" | 35
+
| #1 || [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23119 J23119] || c.Promoter (Strong) || pSB1A2 || class="number" | 35
|-  
|-  
| #2 || J23118 || c.Promoter (Medium) || BBa_J61002 || class="number" |35
| #2 || J23118 || c.Promoter (Medium) || BBa_J61002 || class="number" |35

Revision as of 18:53, 5 October 2011

Parts List

Each part is represented by s number (e.g. #20 for lac promotor) in this notebook. By pointing the mouse on the number within notebook entires, you can find out the details of the part.


Table 1. List of iGEM parts used in our project
Number Reg.ID 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 Notebook

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

Please enable Javascript to view this calendar.

May

'11/05/18 (Wed)

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

'11/05/24 (Tue)

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

'11/05/25(Wed)

  • Making TB(pH=6.7), LB plate

'11/05/26(Thu)

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

'11/05/31(Tue)

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

June

'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

July

'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)

August

'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

September

'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

'11/09/15(Thu)

  • Colony PCR(#19, #36-3-14-5-2-3-17-5-20-3-37-5 (failed), #20-3-29-5)
  • Miniprep(#20, #20-3-14-5-2-3-17-5, #3-14-5-2-3-17-5, #3-17-5)
  • Gel extraction
    • #3-14-5(failed), #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
  • Making WT competent cell
  • Making LB plates(amp:39, cm:18)
  • Culture for Miniprep(#3-11-5 (pSB1C3), #20-3-29-5, #19)
  • Ligation
    • sulAp(ES)-#3-14-5-2-3-17-5_XP-pSB1C3_PCR digest(EP)
    • recNp(ES)-#3-14-5-2-3-17-5_XP-pSB1C3_PCR digest(EP)
    • #36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1C3_PCR digest(EP)
    • #3-27-5_XP-#20_SP
    • #3-17-5_EP-pSB1C3_PCR digest(EP)
    • #3-14-5_EP-pSB1C3_PCR digest(EP)
    • #3-14-5-2-3-17-5_EP-pSB1C3_PCR digest(EP)
    • #20-3-14-5-2-3-17-5_EP-pSB1C3_PCR digest(EP)
  • Culture for Dual Luciferase Assay(#20-3-14-5-2-3-17-5)
  • TLC check (LB, M9)
  • Cell Diffusion Assay
  • Racing Assay (0.25% Agar, 10mM Asp Sol.)
  • Fumaric Acid (Fum) Sol. preparation (failed!!)
  • Sequencing

'11/09/16(Fri)

  • Colony PCR
    • sulAp(ES)-#3-14-5-2-3-17-5_XP-pSB1C3_PCR digest(EP),
    • recNp(ES)-#3-14-5-2-3-17-5_XP-pSB1C3_PCR digest(EP),
    • #36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1C3_PCR digest(EP),
    • #3-27-5_XP-#20_SP,
    • #3-17-5_EP-pSB1C3_PCR digest(EP),
    • #3-14-5_EP-pSB1C3_PCR digest(EP),
    • #3-14-5-2-3-17-5_EP-pSB1C3_PCR digest(EP),
    • #20-3-14-5-2-3-17-5_EP-pSB1C3_PCR digest(EP)
    • #36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1C3_PCR digest(EP) (retry; non-red colonies)
  • Miniprep(#3-11-5 (pSB1C3), #20-3-29-5, #19)
  • PCR amplification(#3-27-5, #36-3-14-5-2-3-17-5, pSB1C3)
  • Sequencing prep(#20-3-29-5)
  • Making agarose gel, WT competent cell,
  • Electrophoresis check(pSB1C3_PCR)
  • Digest(pSB1C3_ES, pSB1C3_EP, #3-27-5_XP, #36-3-14-5-2-3-17-5_ES)
  • Dual luciferase assay
    • #20-3-14-5-2-3-17-5 (1h, 1.5h culture)(IPTG 0, 1, 10, 100uM)(2.5mL)
  • Fumaric Acid (Fum) Sol. preparation
    • TLC test
    • Asp diffusion test
    • Asp racing assay

'11/09/17(Sat)

  • Colony PCR (retry)
    • #3-14-5, #3-17-5 (pSB1C3)
    • #36-3-14-5-2-3-17-5-20-3-37-5
  • Culture for Miniprep(#3-14-5, #3-17-5 (pSB1C3))
  • Ligation(sulAp-pSB1C3, recNp-pSB1C3, #20-#3-27-5)
  • Transformation(#20-3-29-5 into cheZ-)
  • Making medium(.25% Agar. M9& LB each 20 plates)
  • Cell diffusion assay

'11/09/18(Sun)

  • Colony PCR & Culture for Miniprep(#36-3-14-5-2-3-17-5-20-3-37-5, #33-3-14-5-2-3-17-5)
  • Miniprep(#3-14-5, #3-17-5 (pSB1C3))
  • Ligation(#20-#3-27-5, recNp-#3-14-5-2-3-17-5, sulAp-pSB1C3, recNp-pSB1C3, #31-pSB1C3)
  • Cell diffusion assay
    • Innoculate colony(WT / cheZ-/- / cheZ-/- + cheZ plasmid)
  • GFP imaging assay(Culture cells expressing GFP)

'11/09/19(Mon)

  • Culture for Dual luciferase assay(#36-3-14-5-2-3-17-5-20-3-37-5, #33-3-14-5-2-3-17-5, #1-3-14-5-2-3-17-5, #20-3-14-5-2-3-17-5)
  • Miniprep(#36-3-14-5-2-3-17-5-20-3-37-5, #33-3-14-5-2-3-17-5)
  • Ligation(#24-#3-14-5-2-3-17-5, #20-#3-27-5)
  • Sequencing(#36-3-14-5-2-3-17-5-20-3-37-5, #33-3-14-5-2-3-17-5, #20-3-29-5)
  • Dual luciferase assay(#1~, #20~, #36~, #33~ )
  • Asp racing assay(Innoculate colonies)
  • GFP imaging assay

'11/09/20(Tue)

  • colony PCR(20-3-27-5, #24-3-14-5-2-3-17-5)
  • Gel extraction(#3-27-5_XP)
  • PCR amplification -> Digest(#29_EP, #20-3-29-5_EP, #3-29-5_EP)
  • Digest(#20_SP)
  • Sequencing prep.
  • Ethanol precipitation (10ul TE)
    • #3-11-5(pSB1C3): 144.5ng/uL
    • #3-14-5: 728.0ng/uL
    • #3-17-5(pSB1C3): 505.7ng/uL
  • Ligation(#31_EP-pSB1C3_EP, #36-3-29-5_ES-#20-3-37-5_XP-pSB1C3_EP)
  • Luciferase assay
  • Asp diffusion assay

'11/09/21(Wed)

  • Making plates
  • Gel extraction(#20_SP, #29_EP, #3-29-5_EP, #20-3-29-5_EP)
  • Ligation
    • #20_SP-#3-27-5_XP, #24_SP-#3-14-5-2-3-17-5_XP, #29_EP-pSB1C3_EP, #3-29-5_EP-pSB1C3_EP, #20-3-29-5_EP-pSB1C3_EP, #3-14-5-2-3-17-5_EP-pSB1C3_EP
  • Transformation
    • #36 from BioBrick? part
    • #33-3-14-5-2-3-17-5 into BL21 comp.
  • Digest(#3-14-5-2-3-17-5_SP, #3-14-5-2-3-17-5_ES, #3-29-5_ES)
  • Luciferase Assay(#36-3-14-5-2-3-17-5)
  • Cell diffusion assay

'11/09/22(Thu)

  • Ligation (retry)
    • #3-27-5_XP-#20_SP, #3-14-5-2-3-17-5-#24_SP, #29_EP-pSB1C3_EP, #3-29-5_EP-pSB1C3_EP, #20-3-29-5_EP-pSB1C3_EP, #3-14-5-2-3-17-5_EP-pSB1C3_EP
  • Colony PCR(#36(->culture for mini prep), #33-3-14-5-2-3-17-5(->Maybe the amplification was failed))
  • Gel extraction(#3-14-5-2-3-17-5_SP, _ES; #3-29-5_ES)
  • Making plates
    • M9 0.25% Agar. +IPTG +Amp.: 20
    • LB Cm.: 20
  • Ligation
    • #3-14-5-2-3-17-5_SP-#20-3-37-5_XP,
    • #3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1C3,
    • #3-29-5_ES-#20-3-37-5_XP-pSB1C3_EP

'11/09/23(Fri)

  • colony PCR
  • Column purification
  • Ligation
  • PCR amplification
  • Competent cell(BL21(DE3))
  • Asp diffusion test
  • Cell diffusion assay
  • Asp chemotaxis assay

'11/09/26(Mon)

  • Ligation(#20-#3-27-5, #24-#3-14-5-2-3-17-5, recNp-3-14-5-2-3-17-5)
  • Digest(pSB1C3_ES)
  • Miniprep(#3-29-5-pSB1C3, #20-3-29-5-pSB1C3, #36)
  • PCR amplification(pSB1A3, #3-29-5)
  • Ethanol precipitation(#3-29-5-pSB1C3, #20-3-29-5-pSB1C3)

'11/09/27(Tue)

  • Colony PCR(#24-3-14-5-2-3-17-5, #20-3-27-5, #35-3-14-5-2-3-17-5)
  • Digest(#3-29-5_ES, #36_SP, pSB1C3_ES, pSB1C3_XP)
  • Ligation(#20-3-37-5_XP-#3-14-5-2-3-17-5_SP, pSB1C3_ES, #31_EP-pSB1C3_EP)
  • Gel digestion(#3-29-5_ES, #36_SP, pSB1C3_ES, pSB1C3_XP)
  • Nanodrop(#36_SP, pSB1C3)
  • PCR amplification(J04450)
  • Gel extraction(pSB1C3_ES)

'11/09/28(Wed)

  • Nanofrop
    • From PCR : #3-29-5, pSB1C3
    • From Miniprep : #3-29-5, #20-3-27-5, J04450
  • Transformation(#3-5-3-14-5-2-3-17-5) -> BL21(DE) comp.
  • PCR amplification and Digest(EPcut) (#31, #20-3-27-5)
  • Digest(pSB1C3_EP, pSB1C3_XP, #3-29-5_ES, #24_SP_SP, #24_SP)
  • Sequencing(#20-3-27-5, #3-29-5, #3-14-5, #3-17-5)
  • Making AspA culture
  • Cell diffusion assay
  • Asp taxis assay
  • Gel extraction and Nanodrop(pSB1C3_EP, pSB1C3_XP, #3-29-5_ES, #24_SP)
  • Ligation
    • #3-29-5_ES-#20-3-37-5_XP-pSB1C3_EP
    • #3-14-5-2-3-17-5_XP-pSB1C3_XP
    • #20-3-37-5_XP-pSB1C3_XP
    • #3-27-5_XP-pSB1C3_XP
    • #29_XP-pSB1C3_XP
    • #24_SP-#3-14-5-2-3-17-5_XP
    • #36_SP-#3-29-5_XP

'11/09/29(Thu)

  • Ligation(#20-3-27-5_EP-pSB1C3_EP)
  • Gel extraction and Nanodrop(#31_EP, #20-3-27-5_EP)
  • PCR amplification(pSB1C3, #3-14-5-2-3-17-5)
  • Digest(pSB1C3_EP, #3-14-5-2-3-17-5_XP)
  • Colony PCR, PCR amplification, Miniprep and Colony diffusion assay(#36_SP-#3-29-5_XP)
  • Colony PCR and Miniprep(#24-3-14-5-2-3-17-5)

'11/09/30(Fri)

  • Digest(#36-3-29-5_ES)
  • PCR amplification and Digest(EPcut)(#3-14-5-2-3-17-5, #20-3-27-5, #29, #31)
  • Digest(#3-14-5-2-3-17-5_EP, #36-3-29-5_ES, pSB1C3_EP, pSB1C3_ES)
  • Culture for MIniprep from plate stock(#3-14-5-2-3-17-5)
  • Making competent cell(JM109)
  • Gel extraction and Nanodrop(#36-3-29-5_ES_PCR, #36-3-29-5_ES_Miniprep, #3-14-5-2-3-17-5_EP, pSB1C3_EP, pSB1C3_ES, #3-14-5-2-3-17-5_EP, #20-3-27-5_EP, #29_EP, #31_EP)
  • Ligation(#3-14-5-2-3-17-5_EP-pSB1C3_EP, #20-3-27-5_EP-pSB1C3_EP, #29_EP-pSB1C3_EP, #31_EP-pSB1C3_EP, #36-3-29-5_ES-#20-3-37-5_XP-pSB1C3_EP, recNp_ES-pSB1C3_ES, sulAp_ES-pSB1C3_ES, #3-14-5-2-3-17-5_EP-pSB1C3_EP)
  • Sequencing prepartion(#20-3-27-5_F, #36-3-29-5
  • Miniprep, Digest(EXcut) and Nanodrop(#3-14-5-2-3-17-5)
  • Culture for Miniprep(pSB1C3, #36-3-29-5)
  • Making plate LB Cm

October

'11/10/01(Sat)

  • Part Sequencing for parts shipping
  • Miniprep(#36-3-29-5)
  • Sequencing preparation and Colony PCR(#20-3-27-5_F, #36-3-29-5, #24-3-14-5-2-3-17-5)
  • Colony PCR(#3-14-5-2-3-17-5)
  • second colony PCR

'11/10/02(Sun)

  • PCR amplification(using Quick Taq), Digest(Epcut), Gel extraction and Nanodrop(#3-14-5-2-3-17-5)
  • Gel extraction(#3-14-5-2-3-17-5_EX)
  • Dual Luciferase assay(#24)
  • #36-3-29-5 Assay
  • #36-3-29-5-20-3-37-5 Assay
  • Ligation(recNp_ES-#3-14-5-2-3-17-5_EX, #3-14-5-2-3-17-5_EP-pSB1C3_EP)
  • Making agarose gel
  • Asp degradation assay

'11/10/03(Mon)

  • Miniprep, Nanodrop and PCR sequencing(#20-3-27-5, #29, #31, #36-3-29-5-20-3-37-5, recNp, sulAp)
  • Colony PCR(#3-14-5-2-3-17-5-pSB1C3)
  • Preparation for shipping

'11/10/04(Tue)

  • Preparation for shipping(#3-11-5, #3-14-5, #3-17-5, #3-14-5-2-3-17-5, #20-3-27-5, #29, #3-29-5, #20-3-29-5, #36-3-29-5-20-3-37-5, #31, #33, #35)