'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)

'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

'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