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iGEM UT-Tokyo

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'11/5/18 (Wed)

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

'11/5/24 (Tue)

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

'11/5/25(Wed)

• Making TB(pH=6.7), LB plate

'11/5/26(Thu)

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

'11/5/31(Tue)

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

'11/06/01(Wed)

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

'11/06/02(Thu)

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

'11/06/07(Tue)

• Nanodrop
• Transformation
• Culture from frozen stock

'11/06/08(Wed)

• Picking up colony

'11/06/09(Thu)

• Miniprep

'11/06/13(Mon)

• Planting Negative control
• Making frozen stock
• Transformation(BBa_E0240,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)

'11/08/01(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回復培養),#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 誘導(#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(#27, #28, cheZ, lexA, #3-11-5(1), #23,#30 XPcut,#6 EScut,#7 EXcut,#7,#20 SPcut)
• 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(#6 EScut, #7 EXcut, #1,#2,#3,#7,#20 SPcut, #23 XPcut）
• 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 O.N.

'11/08/16(Tue)

• Colony PCR(1:#3-#14-5, 2:#24-#3-17-5, 4:#20-#28, 5:#3-#27, 6:#3-#29, 7:#3-23-#5)
• Culture (4:#20-#28, 14:#5, 16:#14, 17:#17, 18:#2-9, #2-4, #2-3-17-5, #1, #2)
• Incubation (3:#24-#3-11-5, 8:#33-pSB1AK3, 9:#34-pSB1AK3, 10:#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(12:30
• Ligation retry(1:#3-#14-5, 5:#3-#27, 6:#3-#29, N.C.:#3)
• Making Frozen stock(#14、#17)
• Culture test(#14 on 0.3%Agar.(km))(17:10
• 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) -> 21:00 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 )

◦ uvrBp, cheZ from Genome ◦ uvrA from nested-region

• 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,#33 SPcut, #14-5 XPcut, #33 Ecut, #3-27 EScut, pSB1K3 EPcut)
• 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) →O.N.
• 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 (O.N. in 37℃ incubator)

'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, #2, #2-3-11-5 SPcut, #2-3-17-5, cheZ XPcut, #2-3-11-5 EPcut, 8/18uvrBp, 8/17uvrBp, uvrAp EScut)
• Colony PCR retry(#3-29, #34-pSB1AK3)
• Making Frozen Stock
• Check the list of frozen stock(there were too many mistakes)
• 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(1000ml)
• 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 O.N.
  • #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 N.C)

'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 XPcut)
• Ligation (retry) (using today's competent cells & 0802 ⊿cheZ 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 FS)
• Waking from FS (WT, ΔcheZ)

'11/08/28(Sun)

• Making ⊿cheZ comp., LB amp plates, TB
• Colony PCR & master plate preparation -> Failure!!
• Gel extraction (#29 XPcut, #5 EXcut)
• Miniprep & 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(251.3ng/ul),#28(199.7ng/ul)
• Miniprep
  • #20(128.1ng/ul), #14-5(48.3ng/ul)
• 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 reagents and medium
  • 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

'11/9/12 (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)