Team:UT-Tokyo/LabNote

From 2011.igem.org

Revision as of 17:13, 2 October 2011 by Hiro3726 (Talk | contribs)

Parts List

Each part is represented by s number (e.g. #20 for lac promotor) in this notebook. We have also used this number in our hand-written lab notebook. By pointing the mouse on the number in the construct, 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 Diary

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

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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

'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*Miniprep(#5*Dissolution(#1, #2, K16500, #24

'11/06/21(Tue)

*Making frozen stock(#3, B0015, B0040, #9*Passafe culture(#9

'11/06/22(Wed)

*Miniprep(#9

'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

'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

'11/07/06(Wed)

  • Gel extraction (pre)(BBa_I712052)

*Picking up colony(Ba_J23119, #2, #24, #10

'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

'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*Picking up colony(#11*Gel extraction(B#3, #5*Digest (#2

'11/07/14(Thu)

*Miniprep(#1, #11, #20, #21, #22, #23*Electrophoresis(#2*Gel extraction(#2, #3, #5*Passafe(#11

'11/07/15(Fri)

*Ligation(#2-#9, #2-#3, #14-#5, #17-#5*Transformation(#2-#9, #2-#3, #14-#5, #17-#5*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**SPcut : #20, #2-3**XPcut : #10, #11, #14-5, #17-5

'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**SPcut : #20**XPcut : #10, #11, #14-5, #17-5*Transformation(#27, #28

'11/07/25(Mon)

*Colony check(#2, #2-9*Gel Extraction(#20_SP, #10_XP, #11_XP, #14-5_XP, #17-5*Ligation and Transformation(#20-#9, #3-#11, #3-#14-5, #3-#17-5

'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*Ligation(#3-#14-5(Re), #3*Transformation(#15, #27, #28, #30, #3-14-5(Re), #3

'11/07/27(Wed)

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

'11/07/28(Thu)

*Gel extraction(#14-5_XP, #22_SP, #23**SPcut : #1,#2, #3, #24**EScut : #3-11**XPcut : #3-17-5*Ligation(#3-#14-5

'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*Miniprep(#30

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*Gel extraction(#3, #9, #30*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*PCR_2nd(lexA, cheZ, #27, #28*Digest (#30

'11/08/04(Thu)

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

'11/08/05(Fri)

*Digest(#14-5(2) XPcut, #3-11-5*Cloning(PCR)(#27, #28

'11/08/08(Mon)

*Defrost Frozen stock, Miniprep, Nanodrop and Electrophoresis(#5, #20, #23, #30**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

'11/08/09(Tue)

*Miniprep, Electrophoresis and Digest(#5, #14-5(2), #3-11-5*Gel extraction and Nanodrop(#6, #27, #8, cheZ, lexA③, #3-11-5(1), #23, #7_EX, #20, #7*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

'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**EScut : #6**EXcut : #7**SPcut : #1, #2, #3, #7, #20**XPcut : #23

'11/08/11(Thu)

*Gel extraction(#30, #14-5(insert,vector), #5, #3-11-5*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**8/11 PCR product：#27, #28**Miniprep product：#3-14-5(2), #5*Digest（#5 EXcut, #14**8/11 digest product : #5, #14-5, #3-30**8/11 PCR product : #27, #28*Colony PCR (re) (#13-14-5*Miniprep(ligation product : #6-5, #3-23**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**EScut: #3-11-5*Gel extraction(from digest products XPcut: cheZ, uvrBp(PCR products) EScut: #3-11-5*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--*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-*Digest(SPcut: #3, Ecut:#3*Ligation retry(#3-#14-5, #3-#27, #3-#29, Negative control :#3*Frozen stock(#14, #17*Culture test(#14*Miniprep(Ligation products : #2-3-17-5, #14, #17, #1, #2*Waking from frozen stock(#3

'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*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*Culture from 0809 master-plate ⑪ (#2-3-11-5*Gel extraction(8/17#3_SP,#33_SP, #14-5_XP, #33_E, #3-27*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*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-

'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*Colony PCR retry(#3-29, #34-*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 frozen stock(#1, #2, #2-3-17-5(2), #3-30) #7**From ligation products(#7, #3-14-5, #3-27-5, #34, #33-3-11-5**From Frozen Stock (#7_EX, #30_XP, #2-3-17-5_XP, #3-30**From Master plate(#3-14-5_XP, #34-pSB1AK3_SP, #3-27-5*Sulvage #33**From Master plate(#24-3-17-5, #14-5

'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**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**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*Digest (#14-5 XPcut, #3-27-5 Xcut, Pcut (check), #3-27-5*Ligation (#14-#5, #3-30-#5, #3-#30, #34-#3-11-5 #20-#28*#33

'11/08/24(Wed)

*Gel extraction & Electrophoresis(#3-27-5_ES, #14-5_XP, #3-27-5 PCR clean-up system -> digest **#27 XP cut **#28 XP cut *Making plate and amp stock　->4℃ **amp 32 **km 11 (km conc. = 3/4 * that it was) *Miniprep and Nanodrop **#33transfromation *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

'11/08/25(Thu)

*Renilla luciferase assay (with #2-3-17-5*Gel extraction (#27, #28*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*Digest (#14*Passage(#2-3-17-5

'11/08/26(Fri)

*Renilla luciferase assay (with #2-3-17-5*Colony PCR (#33*Gel extraction (#14*Digest (#5*Passage in a shaker(#2-3-17-5

'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*Miniprep and frozen stocks (#33, #14-5*Digest (#20 SPcut, #14-5*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*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*EP for MP check(#14-5, #20*Ligation(#3-#29, pSB1AK3-#2-3-11-5 on Amp/Km plate, #3*Transformation(#2-9 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*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**Plasmid isolation(#34-3-17-5**Part cloning(#36, #37**Titer check(#3-17-5(Takara comp.,1:9gradient), #2-3-11-5(ΔcheZ comp.0802, 1:9gradient), #2-3-11-5

September

'11/09/01(Thu)

  • Colony PCR

**#34-3-17-5(MP), #3-14, #3-29, #35-pSB1AK3, #3-14-5, #37**#27_XP ,#28_XP(0830); #27_XP, #28_XP, #3-30_XP -> failed!, #14-5*Miniprep(#3, #20**#3-14, #3-29, #3-14-5**#3 -> Failed (Mixed with DMSO!), #20**#3-14, #3-29, #3-14-5**#3-27, #20-#3-17-5**#36**Titer check(MP product #3 into 0901 WT comp., MP product #20*Mobility assay(#14-5

'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*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 PCR products were OK but all purified products were lost. **#20-3-17-5, #3-27**#3-14, #3-14-5, #3-29**#20-3-17-5, #3-27, #3-14-5, #3-29**#3_SP, #20_SP, #37_ES, #3-14_ES, #3-29_ES, #3-14-5**#20-3-17-5_XP, #3-27_ES, #3-14-5_XP, #3-29

'11/09/03(Sat)

  • Gel extraction
  • Ligation & Transformation

**#20-#3-14-5, #2-#3-14-5, #3-27-#5**#3-27-#5, #37-#5*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**#2-#3-14-5, #3-27-#5, #3-29-#5, #37-#5, #20-#28**#36**#2, #5*Culture for Miniprep(#20-3-14-5, #20*Digest(#20

'11/09/05(Mon)

*Colony Diffusion Assays (#2-9*Gel extraction(#20**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*Miniprep(#20-3-14-5, #37-5, #36 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**#3-27-#5, #3-30-#5, #2-3-11-5-**#5*Culture for GFP live-imaging (#2-9 4℃ storage *Ninhydrin sol. preparation (Not dissolvable!) -> 4℃ storage

'11/09/07(Wed)

*Colony PCR(#3-27-5, #3-30-5*Digest(#36*Culture from master plate(#36, #3-29-5, #37-5*Miniprep(#36, #3-29-5, #37-5, #3-27-5*Gel extraction(#3-29-5_XP, #37-5_ES, pSB1AK3_EP, #36*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*Culture for Miniprep(#2, #34, #36, #3-27-5, #3-14-5, #2-3-17-5**#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

'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*Miniprep(#2, #34, #36, #2-3-17-5, #3-27-5, #3-14-5**PCR product: #35_SP, #31_XP, #3-14-5_XP, #3-27-5_XP, #3-29-5_XP, #37-5**Miniprep product: #2_SP, #34_SP, #36_SP, #2-3-17-5_EX, #3-14-5*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**PCR products: #34-3-14-5_ES, #36-3-14-5_ES, #20-3-14-5

'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**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**Miniprep product: #3-11-5**PCR-purified product: #3-14-5_EP, #3-17-5_EP, #31_EP, #3-37-5_XP, #36-3-29-5*Miniprep(#34-3-14-5, #36-3-14-5, #20-3-29-5, #2-3-11-5*Gel extraction(#34-3-14-5_ES, #36-3-14-5_ES, #20-3-14-5_ES, #3-11-5**#3-11-5_EP-pSB1C3_EP, #29**#36-3-14-5_ES-#2-3-17-5_EX, #20_SP-#3-27-5**#2-3-11-5**#20-3-29-5**#20-3-29-5*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*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*Colony PCR(#36-3-14-5-#2-3-17-5*Ligation(#20_SP-#3-37-5

'11/09/11(Sun)

  • Digest from Miniprep products

**#3-14-5-2-3-17-5**#36-3-14-5_SP, #20 O.N. *Colony PCR **#3-11-5 (pSB1C3), #29**#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**From Master plate : #20-3-37-5, #29**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**i.PCR amplification -> Digest for >1h -> Heat kill for 20min (#20-3-37-5_XP, #36-3-14-5-2-3-17-5**ii.Digest for >1h -> Heat kill for 20min (#36-3-14-5-2-3-17-5_SP, pSB1A3_EP, #2-3-17-5**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**#20-3-29-5**#20-3-29-5

'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**#36-3-14-5-2-3-17-5-#20-3-37-5-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-**From master plate : #20-3-37-5**From 09/11 Miniprep product : #36-3-14-5-2-3-17-5Note: Only one #20-3-37-5*Miniprep(#20, #29 (pSB1AK3), #2-3-17-5, #2-3-37-5**From PCR : #36-3-14-5-2-3-17-5_ES, #20-3-37-5**From Miniprep : #20-3-37-5**J04450_EP, *#2-9_XP, #20-3-29-5**#20_SP, #36-3-14-5**From OverNight digests : #2-3-17-5**#36-3-14-5-2-3-17-5**From today's PCR digests : #20-3-37-5*Culture for Miniprep(#20-3-37-5*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-**#36-3-14-5-2-3-17-5_ES-#20-3-37-5**#36-3-14-5-2-3-17-5_ES-#20-3-37-5**#3-27-5_XP-#20**#36-3-29-5_ES-#20-3-37-5

'11/9/13 (Tue)

  • Colony PCR

**#36-3-14-5-2-3-17-5_ES-#20-3-37-5**#36-3-14-5-2-3-17-5_ES-#20-3-37-5**#3-27-5_XP-#20**#36-3-29-5_ES-#20-3-37-5**#36-3-14-5-2-3-17-5-20-3-37-5**#20-3-14-5-2-3-17-5**#1-3-14-5-2-3-17-5 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**#20-3-37-5**#20-3-29-5**#2-9Note: #20-3-29-5**#1-3-14-5-2-3-17-5**#20-3-14-5-2-3-17-5**#36-3-14-5-2-3-17-5-20-3-37-5 (#2-3-11-5**#20-3-37-5**#20-3-29-5**#3-27-5**#31**#20**#3-29-5**#20_SP-#3-27-5**#3-14-5-2-3-17-5**#1-3-14-5-2-3-17-5**#20-3-14-5-2-3-17-5**#20-3-37-5

'11/09/14(Wed)

  • Colony PCR

**#3-27-5_XP-#20**#20-3-14-5-2-3-17-5**#1-3-14-5-2-3-17-5**#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**#20**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*Part cloning(#19**#20_SP-#3-29-5**#36-3-14-5-2-3-17-5_ES-#20-3-37-5**#1-3-14-5-2-3-17-5**#20-3-14-5-2-3-17-5**①F⑤R#20-3-37-5**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**#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*Culture for Miniprep(#3-11-5 (pSB1C3), #20-3-29-5, #19**sulAp(ES)-#3-14-5-2-3-17-5**recNp(ES)-#3-14-5-2-3-17-5**#36-3-14-5-2-3-17-5_ES-#20-3-37-5**#3-27-5_XP-#20**#3-17-5**#3-14-5**#3-14-5-2-3-17-5**#20-3-14-5-2-3-17-5*Culture for Dual Luciferase Assay(#20-3-14-5-2-3-17-5

'11/09/16(Fri)

  • Colony PCR

**sulAp(ES)-#3-14-5-2-3-17-5**recNp(ES)-#3-14-5-2-3-17-5**#36-3-14-5-2-3-17-5_ES-#20-3-37-5**#3-27-5_XP-#20**#3-17-5**#3-14-5**#3-14-5-2-3-17-5**#20-3-14-5-2-3-17-5**#36-3-14-5-2-3-17-5_ES-#20-3-37-5*Miniprep(#3-11-5 (pSB1C3), #20-3-29-5, #19*PCR amplification(#3-27-5, #36-3-14-5-2-3-17-5*Sequencing prep(#20-3-29-5*Digest(pSB1C3_ES, pSB1C3_EP, #3-27-5_XP, #36-3-14-5-2-3-17-5**#20-3-14-5-2-3-17-5

'11/09/17(Sat)

  • Colony PCR (retry)

**#3-14-5, #3-17-5**#36-3-14-5-2-3-17-5-20-3-37-5*Culture for Miniprep(#3-14-5, #3-17-5*Ligation(sulAp-pSB1C3, recNp-pSB1C3, #20-#3-27-5*Transformation(#20-3-29-5

'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*Ligation(#20-#3-27-5, recNp-#3-14-5-2-3-17-5, sulAp-pSB1C3, recNp-pSB1C3, #31-

'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

'11/09/20(Tue)

*colony PCR(20-3-27-5, #24-3-14-5-2-3-17-5*Gel extraction(#3-27-5*PCR amplification -> Digest(#29_EP, #20-3-29-5_EP, #3-29-5*Digest(#20**#3-11-5**#3-14-5**#3-17-5*Ligation(#31_EP-pSB1C3_EP, #36-3-29-5_ES-#20-3-37-5

'11/09/21(Wed)

  • Making plates

*Gel extraction(#20_SP, #29_EP, #3-29-5_EP, #20-3-29-5**#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**#36**#33-3-14-5-2-3-17-5*Digest(#3-14-5-2-3-17-5_SP, #3-14-5-2-3-17-5_ES, #3-29-5*Luciferase Assay(#36-3-14-5-2-3-17-5

'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*Colony PCR(#36(->culture for mini prep), #33-3-14-5-2-3-17-5Maybe the amplification was failed)) *Gel extraction(#3-14-5-2-3-17-5_SP, _ES; #3-29-5**#3-14-5-2-3-17-5_SP-#20-3-37-5**#3-14-5-2-3-17-5_ES-#20-3-37-5**#3-29-5_ES-#20-3-37-5

'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*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-


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