2010.08.17

• plasmid extraction of aptamer on plasmid and digestion aptamer SP
  • digestion protocol

2010.08.18

• gel extraction >> purification >> ligation >> transformation of aptamer(SP)
  • Transformation failed, so we digest again on 8/20. (no colonies)
  • ligation protocol

2010.08.20

• digestion of aptamer(SP) and terminator(XP)
  • Do gel extraction, and when running electrophoresis we found that digestion failed so we restart to digest again.

2010.08.21

• gel extraction and purification of aptamer(SP)
  • We checked the result of digestion and aptamer succeed but terminator failed.
  • continued to purify aptamer(SP), and ligation aptamer(SP) and terminator(XP,from SSrA)
  • transformation

2010.08.22

• 3 in 1 of aptamer+terminator
  • colony PCR and run electrophoresis
  • negative was contaminated

2010.08.23

• colony PCR of aptamer+terminator and plasmid extraction
  • check result of 8/22 but contaminated
  • restart to digest terminator(XP) and aptamer+terminator(XP)

2010.08.24

• aptamer purification and eIF4A PCR
  • We run electrophoresis to check and terminator(XP) succeed, aptamer+terminator(XP) failed.
  • eIF4A PCR(use tag)

2010.08.25

• 3 in 1 of aptamer and eIF4A electrophoresis(and design primer)

2010.08.26

• plasmid extraction,digestion and purification of apt.+ter.
  • result of extraction → 3.4.5 be poluted,so use 1.2. for digest
  • digestion protocol(apt.+ter.)(XP)

Total 20μl DNA 16μl Digestion buffer 2μl Enzyme 1 (X) 1μl Enzyme 2 (P) 1μl electrophoresis of tag Gradient(eIF4A) and apt.+ter. left → tube 1. right → tube 2. the result of check → It is possible for VR.VF2 to be polluted. also purification of RBS PCR of complete eIF4A → purification→ digestion(XP)→ ligation with pSB1A2(XP) → transformation real PCR solution of complete eIF4A(X2)(take out at 18:50) Total: 50μl X2μl FP RP Template Template 1μl negative VF2 VR FP 1μl 2μl positive VF2 VR complete GFP RP 1μl 2μl dNTP 4μl 8μl 10XBuff. 5μl 10μl pfu 1μl 2μl ddH2O 37μl 74μl PCR Protocol 94 120s 94 15s 53.6 20s 72 90s 35 cycles 72 60s ligation protocol(10mins) Total 10μl buffer 1μl ligase 0.5μl DNA1(eIF4A[XP]) 3μl DNA2(pSB1A2[XP]) 1μl ddH2O 4.5μl transform(competent cell 30ul) ligation of [apt.+ter.]+RBS ligation protocol Total 10μl buffer 1μl ligase 0.5μl DNA1(apt.+ter[XP]) 7.7μl DNA2(RBS[SP]) 0.8μl 2010.08.27 colony PCR of apt+ter+RBS colony PCR of eIF4A+pSB1A2 result of both of the PCR failed, it is possible for the negative control to be contaminated. We change the system of colony PCR, and PCR the eIF4A+pSB1A2 again. 2010.08.28 colony PCR check 2010.08.29 plasmid extraction of eIF4A on pSB1A2 and apt.+ter.+RBS digestion of eIF4A on pSB1A2(XP)[check], apt.+ter.+RBS(XP), pLac(SP) and eIF4A(XP) digestion protocol of eIF4A on pSB1A2(XP) and apt.+ter.+RBS(XP) Total 20μl DNA 16μl Digestion buffer 2μl Enzyme 1 (X) 1μl Enzyme 2 (P) 1μl band1~6 → apt.+ter.+RBS(XP) // maker // eIF4A on pSB1A2(XP) // result of check is that (apt.+ter.)+(RBS) did not ligase, so we decided to ligase them again. digestion protocol of pLac(SP) and eIF4A(XP) Total 20μl DNA 10μl Digestion buffer 2μl Enzyme 1 (S) 1μl Enzyme 2 (P) 1μl ddH2O 6μl purification of eIF4A(XP) and pLac(SP) ligation of (apt.+ter/XP)+(RBS/SP) ligation protocol Total 10μl buffer 1μl ligase 0.5μl DNA1(apt.+ter[XP]) 7.7μl DNA2(RBS[SP]) 0.8μl transformation of the above one 2010.08.30 We discover that eIF4A sequence has 2 PstI enzyme cutting sites, so we design primer and make a eIF4A sequence by ourselves. There are no colonies on the transformation plate yesterday(apt.+ter(XP))+(RBS(SP)), so we digest the apt.+ter/XP and RBS/SP for the beginning. digestion protocol of apt.+ter(XP) Total 20μl DNA 16μl Digestion buffer 2μl Enzyme 1 (X) 1μl Enzyme 2 (P) 1μl digestion protocol of RBS(SP) Total 20μl DNA 10μl Digestion buffer 2μl Enzyme 1 (X) 1μl Enzyme 2 (P) 1μl ddddH2O 6μl run gel and gel extraction band1~10 → apt.+ter.(XP) // maker // eIF4A ligation of apt.+ter(XP)+(RBS(SP) transformation 2010.08.31 3 in 1 of apt+term+RBS colony PCR of apt+term+RBS Total: 50μl X2μl FP RP Template Template 1μl negative VF2 VR FP 1μl 5μl positive VF2 VR complete GFP RP 1μl 5μl dNTP 2μl 10μl 10XBuff. 5μl 25μl taq 0.25μl 1.25μl ddH2O 39.75μl 198.75μl PCR Protocol 94 60s 94 15s 55 20s 72 40s 30 cycles 72 300s tube 4 ligase successfully. colony PCR(for test) of eIF4A 1,2,3(mutant part 1.2.3) >>>> 55'C Total: 50μl X2.5μl FP RP Template Template 1μl 5.6μl negative FP 1μl positive eIF4A-split-A-FP eIF4A-split-B-RP eIF4A/C212-2 RP 1μl mutant1 eIF4A-split-A-FP eIF4A-mutant-1-RP eIF4A/C212-2 dNTP 2μl 5μl mutant2 eIF4A-mutant-1-FP eIF4A-mutant-2-RP eIF4A/C212-2 10XBuff. 5μl 12.5μl mutant3 eIF4A-mutant-2-FP eIF4A-split-B-RP eIF4A/C212-2 taq 0.25μl 0.625μl ddH2O 39.75μl 99.375μl PCR Protocol 94 60s 94 15s 53.6 20s 72 80s 30 cycles 72 300s mut.2 and3 didn't have bands, so we PCR test again. colony PCR(for test) of eIF4A 1,2,3(mutant part 1.2.3)>>>>>>>> for the second time! >>>> 51'C 2010.09.01 plasimd extraction of apt.+term.+RBS(X2 tubes) digestion of apt.+term.+RBS(XP) and use our own pLac(SP) for ligation digestion protocol of apt.+term.+RBS(XP) Total 20μl DNA 16μl Digestion buffer 2μl Enzyme 1 (X) 1μl Enzyme 2 (P) 1μl run gel >> gel extraction of apt.+term.+RBS(XP) purification of apt.+term.+RBS(XP)>> ligation >> transformation(9/1 18:00) ligation protocol Total 10μl buffer 1μl ligase 0.5μl DNA1(apt.+ter+RBS[XP]) 6.7μl length:235 DNA2(pLac[SP]) 1.8μl length:200 eIF4A PCR test result(yesterday) all succeed.YA! PCR of eIF4A(pfu) Total: 50μl X2μl FP RP Template length Template 1μl negative FP 1μl positive eIF4A-split-A-FP eIF4A-split-B-RP eIF4A/C212-2 1216 RP 1μl mutant1 eIF4A-split-A-FP eIF4A-mutant-1-RP eIF4A/C212-2 254 dNTP 4μl 8μl mutant2 eIF4A-mutant-1-FP eIF4A-mutant-2-RP eIF4A/C212-2 191 10XBuff. 5μl 10μl mutant3 eIF4A-mutant-2-FP eIF4A-split-B-RP eIF4A/C212-2 811 pfu 1μl 2μl ddH2O 37μl 74μl PCR Protocol 94 120s 94 30s 51 30s 72 160s 35 cycles 72 60s 2010.09.02 3 in 1 of (apt.+term.+RBS)+(pLac) >> PCR >> run gel for check length calculate Total 661 aptamer+scar 58+8 terminator+scar 129+8 RBS+scar 12+8 pLac 200 VR-VF2 238 PCR protocol for test(6tubes) Total: 50μl X3μl FP RP Template Template 1μl negative VF2 VR FP 1μl 3μl positive VF2 VR complete GFP RP 1μl 3μl dNTP 2μl 6μl 10XBuff. 5μl 15μl taq 0.25μl 0.75μl ddH2O 39.75μl 119.25μl PCR Protocol 94 60s 94 15s 55 20s 72 60s 30 cycles 72 300s result all succeed.(tube1 was taken by SsrA group as negative.= = run gel of eIF4A mutant1.2.3 >> gel extraction >> purification >> PCR of mutant1+2 and mutant2+3 run gel result of mutant1.2.3 all succeed. PCR protocol for test(4tubes) Total: 50μl X2μl FP RP Template length Template 1μl negative FP 1μl positive eIF4A-split-A-FP eIF4A-split-B-RP eIF4A/C212-2 1216 RP 1μl mutant1+2 eIF4A-split-A-FP eIF4A-mutant-2-RP eIF4A/C212-2 445-8 dNTP 4μl 8μl mutant2+3 eIF4A-mutant-1-FP eIF4A-split-B-RP eIF4A/C212-2 1002-8 10XBuff. 5μl 10μl pfu 1μl 2μl ddH2O 37μl 74μl PCR Protocol 94 120s 94 30s 51 30s 72 160s 35 cycles 72 600s result all failed.So we do again at 9/3. 2010.09.03 plasimd extraction of aptamer+terminator+RBS+pLac, and the aptamer part have been done!!! (can use any other promotor) PCR of eIF4A mutant1+2, mutant2+3 again >> run gel >> gel extraction >> purification protocol as yesterday. result all succeed. PCR of eIF4A mutant(1+2)+3, mutant1+(2+3) PCR protocol Total: 50μl X2μl FP RP Template length Template 1μl negative FP 1μl positive eIF4A-split-A-FP eIF4A-split-B-RP eIF4A/C212-2 1216 RP 1μl mutant(1+2)+3 eIF4A-split-A-FP eIF4A-split-B-RP mutant(1+2) 1216 dNTP 4μl 8μl mutant1+(2+3) eIF4A-split-A-FP eIF4A-split-B-RP mutant(2+3) 1216 10XBuff. 5μl 10μl pfu 1μl 2μl ddH2O 37μl 74μl PCR Protocol 94 120s 94 30s 51 30s 72 160s 35 cycles 72 600s 2010.09.04 2010.09.05 2010.09.06 2010.09.07 2010.09.08 3 in 1 of pSB1C3+aptamer/+apt+term/+RBS+apt+term/+pLac+RBS+apt+term colony PCR (12 tubes) pSB1C3+RBS+apt+term and pSB1C3+pLac+RBS+apt+term need to be transformed again( Because there are no coloines on plate.) 2010.09.09 elute dried DNA(pGEX-eIF4A) from kind Pro.C.Proud(use 60ul TE buffer and put at 4'C overnight) pGEX is anti ampicillin. plasmid extraction of pSB1C3+aptamer and pSB1C3+pLac run gel of (1) pSB1C3+aptamer+terminator (2) pSB1C3+pLac (3) posotive (4) negative result of the above: all succeed!!So, pSB1C3+pLac can be put on our 2010 biobrick!(LB tube of pSB1C3+aptamer have nothing.) ligation of (pLac+RBS+aptamer+terminator)+pSB1C3 >> transformation ligaiton protocol Total 10μl buffer 1μl ligase 0.5μl DNA1(pSB1C3) 2μl DNA2(pLac+RBS+aptamer+terminator) 3μl ddH2O 3.5μl 2010.09.10 3 in 1 of pSB1C3+pLac+RBS+apt+term and pSB1C3+RBS+apt+term(because LB tube did not be put for cultivating><) ligation of (pSB1C3)+(apt+term) >> transformation Total 10μl buffer 1μl ligase 0.5μl DNA1(pSB1C3) 1μl DNA2(pLac+RBS+aptamer+terminator) 1.5μl ddH2O 6μl PCR colony of (1.2) pSB1C3+pLac+RBS+apt+term (3) pSB1C3+aptamer (4) pSB1C3+RBS+apt+term PCR protocol for test(6tubes) Total: 50μl X3μl FP RP Template Template 1μl negative VF2 VR FP 1μl 3μl positive VF2 VR pLac+RBS+apt+term RP 1μl 3μl dNTP 2μl 6μl 10XBuff. 5μl 15μl taq 0.25μl 0.75μl ddH2O 39.75μl 119.25μl PCR Protocol 94 60s 94 15s 55 20s 72 60s 30 cycles 72 300s 2010.09.11 plasmid extraction of pSB1C3+pLac+RBS+aptamer+terminator and pSB1C3+RBS+aptamer+terminator succeed! Have two new 2010 biobricks! The PCR of yesterday failed. So, do it again. And the result of the PCR today sill failed.(no bands><), we still figure what the problem is. The plate of pSB1C3+aptamer+terminator have no colonies, so ligate again. digestion of pSB1C3(XP) digestion protocol Total 20μl DNA 10μl Digestion buffer 2μl Enzyme 1 (X) 1μl Enzyme 2 (P) 1μl ddH2O 6μl ligation of pSB1C3+aptamer+terminator >> transformation ligaiton protocol Total 10μl buffer 1μl ligase 0.5μl DNA1(pSB1C3) 3.5μl DNA2(aptamer+terminator) 1.5μl ddH2O 3.5μl 2010.09.12 colony PCR of complete eIF4A real PCR protocol(taq) Total: 50μl X1.5μl FP RP Template Template 1μl negative VF2 VR FP 1μl 1.5μl positive VF2 VR complete GFP RP 1μl 1.5μl dNTP 2μl 3μl 10XBuff. 5μl 7.5μl taq 0.25μl 0.375μl ddH2O 39.75μl 596.25μl PCR Protocol 94 60s 94 15s 51 20s 72 80s 30 cycles 72 300s 2010.09.13 transform (pGEX-eIF4A)(because the concentration of template from C.Proud is too low) PCR(3 tubes) Test Total: 50μl X1.5μl FP RP Template length Template 1μl negative FP 1μl positive VF2 VR GFP RP 1μl eIF4A(0.3+water0.2) eIF4A-split-A-FP eIF4A-split-B-RP eIF4A 1216 dNTP 4μl 3μl 10XBuff. 5μl 7.5μl pfu 1μl 0.375μl ddH2O 37μl 59.625μl PCR Protocol 94 60s 94 15s 55 20s 72 80s 30 cycles 72 300s 2010.9.14 3in1 of pGEX-eIF4A colony PCR(6tubes) Total: 50μl X3μl FP RP Template length Template 1μl negative FP 1μl 3μl positive(eIF4A) VF2 VR GFP RP 1μl 3μl pGEX-eIF4A(1) eIF4A-split-A-FP eIF4A-split-B-RP eIF4A 1216 dNTP 2μl 6μl pGEX-eIF4A(2) eIF4A-split-A-FP eIF4A-split-B-RP eIF4A 1216 10XBuff. 5μl 15μl pGEX-eIF4A(3) eIF4A-split-A-FP eIF4A-split-B-RP eIF4A 1216 taq 0.25μl 0.75μl pGEX-eIF4A(4) eIF4A-split-A-FP eIF4A-split-B-RP eIF4A 1216 ddH2O 39.75μl 119.25μl PCR Protocol 94 60s 94 15s 55 20s 72 80s 30 cycles 72 300s 2010.9.15 real PCR(5tubes) of split eIF4A mutI, mutII, mutIII Total: 50μl X2.5μl FP RP Template length Template 1μl negative FP 1μl 2.5μl positive(eIF4A) eIF4A-split-A-FP eIF4A-split-B-RP eIF4A 1216 RP 1μl 2.5μl mutI(1) eIF4A-split-A-FP eIF4A-mutI-RP eIF4A 258 dNTP 2μl 10μl mutII(2) eIF4A-mutI-FP eIF4A-mutII-RP eIF4A 191 10XBuff. 5μl 12.5μl mutIII(3) eIF4A-mutII-FP eIF4A-split-B-RP eIF4A 811 MgSO4 4μl 10μl KOD 1μl 2.5μl ddH2O 39.75μl 82.5μl PCR Protocol 94 120s 94 30s 55 30s 72 160s 35 cycles 72 600s Total: 50μl X2μl FP RP Template length Template 1μl negative FP 1μl 2μl positive(eIF4A) eIF4A-split-A-FP eIF4A-split-B-RP eIF4A 1216 RP 1μl 2μl mutI+mutII eIF4A-split-A-FP eIF4A-mutII-RP mutI+mutII 424 dNTP 2μl 4μl mutII+mutIII eIF4A-mutI-FP eIF4A-split-B-RP mutII+mutIII 983 10XBuff. 5μl 10μl mutI+mutII+mutIII eIF4A-split-A-FP eIF4A-split-B-RP mutI+mutII+mutIII 1216 pfu 0.25μl 0.5μl ddH2O 39.75μl 79.5μl PCR Protocol 94 120s 94 30s 55 30s 72 160s 35 cycles 72 600s 2010.9.16 run gel→gel extraction real PCR real PCR protocol Total: 50μl X2μl FP RP Template length Template 1μl negative FP 1μl 2μl positive(eIF4A) eIF4A-split-A-FP eIF4A-split-B-RP eIF4A 1216 RP 1μl 2μl mut(I+II)+III eIF4A-split-A-FP eIF4A-split-B-RP mutI+mut(II+III) 1216 dNTP 2μl 4μl mutI+(II+III) eIF4A-split-A-FP eIF4A-split-B-RP mutI+mutII+mutIII 1216 10XBuff. 5μl 10μl mut(I+II+III) eIF4A-split-A-FP eIF4A-split-B-RP mutI+mutII+mutIII 1216 pfu 0.25μl 0.5μl ddH2O 39.75μl 79.5μl PCR Protocol 94 120s 94 30s 55 30s 72 160s 35 cycles 72 600s digestion of eIF4A on pGEX digestion protocol Total 20μl DNA(pGEX-eIF4A1) 2μl Digestion buffer 2μl Enzyme 1 (X) 1μl Enzyme 2 (P) 1μl ddH2O 14μl digestion of mut(I+II+III) digestion protocol Total 20μl DNA(mut(I+II)+III) 10μl Digestion buffer 2μl Enzyme 1 (X) 1μl Enzyme 2 (P) 1μl ddH2O 6μl ligation: (1)eIF4A on pSB1C3 (2)mut(I+II)+III ligation protocol Total 10μl buffer 1μl ligase 0.5μl DNA1(eIF4A) 3μl DNA2(pSB1C3) 2μl ddH2O 3.5μl 2010.9.17 PCR of eIF4A(3 tubes) PCR protocol Total: 50μl X1.5μl FP RP Template length Template 1.5μl negative FP 1μl positive(mut(I+II)+III) eIF4A-split-A-FP eIF4A-split-B-RP mut(I+II)+III 1216 RP 1μl mut(I+II)+III eIF4A-FP eIF4A-split-B-RP mutI+mut(II+III) 1216 dNTP 2μl 3μl 10XBuff. 5μl 7.5μl pfu 0.25μl 0.375μl ddH2O 39.75μl 59.625μl PCR Protocol 94 120s 94 30s 55 30s 72 80s 35 cycles 72 420s PCR again same protocol 2010.9.18 2010.9.20 purification of eIF4A(I+II)+III ligation and transformation of eIF4A(I+II)+III and pSB1C3 ligation protocol Total 10μl buffer 1μl ligase 0.5μl DNA1(eIF4A) 3μl eIF4A length 1216 DNA2(pSB1C3) 2μl pSB1C3 length 316 ddH2O 3.5μl PCR of eIF4A-A,eIF4A-B(4tubes) PCR protocol Total: 50μl X2μl FP RP Template length Template 1μl negative FP 1μl positive(eIF4A) eIF4A-split-A-FP eIF4A-split-B-RP c212-2 1216 RP 1μl eIF4A-A eIF4A-split-A-FP eIF4A-split-A-RP mut(I+(II+III)) 1216 dNTP 2μl 4μl eIF4A-B eIF4A-split-B-FP eIF4A-split-B-RP mut(I+(II+III)) 1216 10XBuff. 5μl 10μl pfu 0.25μl 0.5μl ddH2O 39.75μl 79.5μl PCR Protocol 94 120s 94 30s 55 30s 72 80s 35 cycles 72 420s 2010.9.21 PCR of GFP-A,GFP-B,RFP-A,RFP-B(6tubes) PCR protocol Total: 50μl X3μl FP RP Template length Template 1μl negative FP 1μl positive(RFP) VF2 VR RFP 776 RP 1μl GFP-A GFP-split-A-FP GFP-split-A-RP GFP 517 dNTP 2μl 6μl GFP-B GFP-split-B-FP GFP-split-B-RP GFP 298 10XBuff. 5μl 15μl RFP-B RFP-split-B-FP RFP-split-B-RP GFP 508 pfu 0.25μl 0.75μl RFP-B RFP-split-B-FP RFP-split-B-RP GFP 268 ddH2O 39.75μl 119.25μl PCR Protocol 94 120s 94 30s 55 30s 72 80s 35 cycles 72 420s 3 in 1 of eIF4A(I+II)+III and pSB1C3 PCR of eGFP(A)+eIF4A(A),eGFP(B)+eIF4A(B) PCR protocol Total: 50μl X2μl FP RP Template length Template 1μl negative FP 1μl positive VF2 VR eIF4A(eIF4A on pGEX) 1216 RP 1μl system GA GFP-split-A-FP eIF4A-split-A-RP GFP 1195 dNTP 2μl 4μl system GB GFP-split-B-FP eIF4A-split-B-RP GFP 847 10XBuff. 5μl 10μl system RA RFP-split-A-FP eIF4A-split-A-RP GFP 1181 pfu 0.25μl 0.5μl system RB RFP-split-A-FP eIF4A-split-A-RP GFP 818 ddH2O 39.75μl 79.5μl PCR Protocol 94 120s 94 30s 55 30s 72 80s 35 cycles 72 420s 2010.9.22 gel extraction of system GA,system GB,system RA,system RB digestion of eIF4A on pSB1C3,system GA,system GB, system RA,system RB digestion protocol Total 20μl DNA 16μl Digestion buffer 2μl Enzyme 1 (X) 1μl Enzyme 2 (P) 1μl ligation of system GA,system GB,system RA ligation protocol Total 10μl buffer 1μl ligase 0.5μl DNA1(GA or GB) 0.5μl DNA2(pSB1C3) 4.5μl ddH2O 3.5μl ligation of system RB ligation protocol Total 10μl buffer 1μl ligase 0.5μl DNA1(~) 1.5μl DNA2(pSB1C3) 3.5μl ddH2O 3.5μl 2010.9.23 digstion of pSB1C3+pLac(X,P) digestion protocol Total 20μl DNA 16μl Digestion buffer 2μl Enzyme 1 (X) 1μl Enzyme 2 (P) 1μl gel extraction ligation of system GA,system GB,system RA,system RB, ligation protocol Total 10μl buffer 1μl ligase 0.5μl DNA1(~) 2.5μl DNA2(pSB1C3(X,P)) 5μl ddH2O 1μl 2010.9.24 3 in 1 of system GA, system GB, system RA, system RB colony PCR of system GA, system GB, system RA, system RB PCR protocol Total: 50μl X5μl FP RP Template length Template 1μl negative FP 1μl positive(eIF4A on pGEX) eIF4A-split-A-Fp eIF4A-split-B-Rp eIF4A on pGEX 1216 RP 1μl system GA on pSB1C3 GFP-split-A-FP GFP-split-A-RP GFP 1195 dNTP 2μl 10μl system GB on pSB1C3 GFP-split-B-FP GFP-split-B-RP GFP 847 10XBuff. 5μl 25μl system RA on pSB1C3 RFP-split-B-FP RFP-split-B-RP GFP 1181 pfu 0.25μl 1.25μl system RB on pSB1C3 RFP-split-B-FP RFP-split-B-RP GFP 818 ddH2O 39.75μl 198.75μl PCR Protocol 94 60s 94 15s 55 20s 72 80s 35 cycles 72 300s 2010.09.25 plasmid extration of (1)GFP-eIF4A-A-1 (2)GFP-eIF4A-A-2(failed) (3)GFP-eIF4A-B-1 (4)GFP-eIF4A-B-2(failed) (5)RFP-eIF4A-A-1 (6)RFP-eIF4A-A-2 (7)RFP-eIF4A-B-1(failed) (8)RFP-eIF4A-B-2 digestion of GA(XP),GB(XP),RA-1(XP),RA-2(XP),pGEX-e(P),pGEX-e(X) digestion protocol Total 20μl DNA 16μl Digestion buffer 2μl Enzyme 1 (X) 1μl Enzyme 2 (P) 1μl ligation of GA,GB,RA,RB ligation protocol of GA,GB Total 10μl buffer 1μl ligase 0.5μl DNA1(pSB1A2) 2μl DNA2(GA/GB)) 5μl ddH2O 1.5μl ligation protocol of RA,RB Total 10μl buffer 1μl ligase 0.5μl DNA1(pSB1A2) 2μl DNA2(RA/RB)) 1μl ddH2O 5.5μl digestion of pSB1A2(XP), pSB1C3(XP) digestion protocol(X2) Total 20μl DNA 16μl Digestion buffer 2μl Enzyme 1 (X) 1μl Enzyme 2 (P) 1μl 2010.09.26 colony PCR of GA(X2),GB(X2),RA(X2),RB(X2),on pSB1A2 PCR protocol Total: 50μl X6μl length Template 1μl negative FP(VF2) 1μl positive(GFP2) 720+238 RP(VR) 1μl GFP-eIF4A-A-1,2 1387 dNTP 2μl 12μl GFP-eIF4A-B-1,2 1042 10XBuff. 5μl 30μl RFP-eIF4A-A-1,2 1387 tag 0.25μl 1.5μl RFP-eIF4A-B-1,2 1012 ddH2O 39.75μl 238.5μl PCR Protocol 94 60s 94 15s 55 20s 72 90s 30 cycles 72 300s digestion and gel extration of pSB1C3(XP), pSB1A2(XP) 2010.09.27 plasmid extraction of GFP-e-A-2,GFP-e-B-2,RFP-e-A-2,RFP-e-B-2 digestion of GFP-e-A-2,GFP-e-B-2,RFP-e-A-2,RFP-e-B-2 digestion protocol Total 20μl DNA 16μl Digestion buffer 2μl Enzyme 1 (X) 1μl Enzyme 2 (P) 1μl run gel and check.....failed! ligation of GA,GB,RA,RB ligation protocol of GA,GB Total 10μl buffer 1μl ligase 0.5μl DNA1(pSB1A2) 2μl DNA2(GA/GB)) 5μl ddH2O 1.5μl ligation protocol of RA,RB Total 10μl buffer 1μl ligase 0.5μl DNA1(pSB1A2) 2μl DNA2(RA/RB)) 1μl ddH2O 5.5μl transformation. 2010.09.28 3 in 1 of colony PCR of GA,GB,RA,RB PCR protocol Total: 50μl X5μl length Template 1μl negative FP(VF2) 1μl positive(GFP2) 720+238 RP(VR) 1μl GFP-eIF4A-A-1,2 1387 dNTP 2μl 10μl GFP-eIF4A-B-1,2 1042 10XBuff. 5μl 25μl RFP-eIF4A-A-1,2 1387 tag 0.25μl 1.25μl RFP-eIF4A-B-1,2 1012 ddH2O 39.75μl 198.75μl PCR Protocol 94 60s 94 15s 55 20s 72 90s 30 cycles 72 300s 2010.09.29 plasmid extractio of GA(1),GB(2),RA(1),RB(2) digestion of GA(1),GB(2),RA(1),RB(2) digestion protocol Total 20μl DNA 16μl Digestion buffer 2μl Enzyme 1 (X) 1μl Enzyme 2 (P) 1μl 2010.09.30 ligation of GA,GB,RA,RB on pSB1A2 ligation protocol of GA,GB Total 10μl buffer 1μl ligase 0.5μl DNA1(pSB1A2) 2μl DNA2(GA/GB)) 5μl ddH2O 1.5μl ligation protocol of RA,RB Total 10μl buffer 1μl ligase 0.5μl DNA1(pSB1A2) 2μl DNA2(RA/RB)) 1μl ddH2O 5.5μl 2010.10.01 3in1 of GA(2),GB(2),RA(1),J13002(2) (use liquid to replace for the LB buffer) PCR protocol Total: 50μl X4.5μl Template 1μl negative FP 1μl positive(GFP2) RP 1μl dNTP 2μl 9μl 10XBuff. 5μl 22.5μl tag 0.25μl 1.125μl ddH2O 39.75μl 178.875μl PCR Protocol 94 60s 94 15s 55 20s 72 80s 35 cycles 72 300s digestion GeA,GeB,ReA,ReB digestion protocol Total 20μl DNA 16μl Digestion buffer 2μl Enzyme 1 (X) 1μl Enzyme 2 (P) 1μl PCR of GeA,GeB,ReA,ReB PCR protocol Total: 50μl X4μl FP RP Template length Template 1μl negative FP 1μl RP 1μl system GA GFP-split-A-FP eIF4A-split-A-RP dNTP 2μl 8μl system GB GFP-split-B-FP eIF4A-split-B-RP 10XBuff. 5μl 20μl system RA RFP-split-A-FP eIF4A-split-A-RP pfu 0.25μl 1μl system RB RFP-split-B-FP eIF4A-split-B-RP ddH2O 39.75μl 159μl PCR Protocol 94 120s 94 30s 55 30s 72 80s 35 cycles 72 420s 2010.10.02 plasmid extraction of GA, GB, RA, J13002 digestion(XP) to check length purify the PCR yesterday and digest(DNA:16) gel extraciton GA.RB digest again. RUSULT: THe result of digestion from plasmid extraction is wrong. The reusult of PCR is right 2010.10.03 colony PCR of GeA, GeB, ReA, ReB on J13002 PCR protocol Total: 50μl X10μl Template 1μl FP 1μl positive(GFP2) RP 1μl system GA on J13002 dNTP 2μl 20μl system GB on J13002 10XBuff. 5μl 50μl system RA on J13002 pfu 0.25μl 2.5μl system RB on J13002 ddH2O 39.75μl 397.5μl PCR Protocol 94 60s 94 15s 55 20s 72 100s 30 cycles 72 300s ligation of aptamer(XP)+pLac(SP) ligation protocol Total 10μl buffer 1μl ligase 0.5μl DNA1(pLac) 1μl DNA2(aptamer) 3μl ddH2O 4.5μl digestion of J13002(SP) digestion protocol Total 20μl DNA 10μl Digestion buffer 2μl Enzyme 1 (S) 1μl Enzyme 2 (P) 1μl ddH2O 6μl 2010.10.04 purification of J13002(SP) 3in1 of aptamer(XP)+pLAC(SP) colony PCR PCR protocol Total: 50μl X2μl Template 1μl negative FP 1μl positive(GFP2) RP 1μl dNTP 2μl 4μl 10XBuff. 5μl 10μl tag 0.25μl 0.5μl ddH2O 39.75μl 79.5μl PCR Protocol 94 60s 94 15s 55 20s 72 40s 30 cycles 72 300s ligation of J13002(SP)+GA,GB,RA,RB(XP) ligation protocol of GA,GB Total 10μl buffer 1μl ligase 0.5μl DNA1(J13002) 4μl DNA2(~) 1μl ddH2O 3.5μl ligation protocol of RA,RB Total 10μl buffer 1μl ligase 0.5μl DNA1(J13002) 4.5μl DNA2(~) 0.5μl ddH2O 3.5μl transformation 2010.10.06 3in1 of pTet+RBS+GA,GB,RA,RB colony PCR PCR protocol Total: 50μl X6μl Template 1μl FP 1μl RP 1μl dNTP 2μl 12μl 10XBuff. 5μl 30μl pfu 0.25μl 1.5μl ddH2O 39.75μl 238.5μl PCR Protocol 94 60s 94 15s 55 20s 72 100s 30 cycles 72 300s plasmid extraction of aptamer(XP)+pLac(SP)] run gel of pTet+RBS+GA(X5),GB(X4),RA(X4),RB(X4) cultivate LB: GA tube5, GB tube3, RA tube1, RA tube2 2010.10.07 plasmid extraction of pTet+RBS+GA,GB,RA1,RA2 digestion of pTet+RBS+GA,GB,RA1,RA2 digestion protocol Total 20μl DNA 16μl Digestion buffer 2μl Enzyme 1 (X) 1μl Enzyme 2 (P) 1μl digestion of J13002(SP) > purification > ligation of J13002(SP)+RBS(XP) 2010.10.08 purification of pLac+apt(SP) digestion of pSB1C3(XP),pLac+apt(XP) digestion protocol Total 20μl DNA 16μl Digestion buffer 2μl Enzyme 1 (X) 1μl Enzyme 2 (P) 1μl run gel of pSB1C3(XP),pLac+apt(XP),terminator(XP) then gel extraction. PCR of J13002+GB,J13002+RB1,J13002+RB2 PCR protocol Total: 50μl X2μl Template 1μl FP 1μl RP 1μl dNTP 2μl 4μl 10XBuff. 5μl 10μl tag 0.25μl 0.5μl ddH2O 39.75μl 79.5μl PCR Protocol 94 60s 94 15s 55 20s 72 100s 30 cycles 72 300s 3in1 of J13002+GB,J13002+RB1,J13002+RB2 2010.10.09 PCR of J13002+GB,J13002+RB1,J13002+RB2 colony PCR protocol Total: 50μl X2μl Template 1μl FP 1μl RP 1μl dNTP 2μl 4μl 10XBuff. 5μl 10μl tag 0.25μl 0.5μl ddH2O 39.75μl 79.5μl PCR Protocol 94 60s 94 15s 55 20s 72 100s 30 cycles 72 300s plasmid extraction of J13002+GB,J13002+RB1,J13002+RB2 digestion of J13002+GB,J13002+RB1,J13002+RB2 digestion protocol Total 20μl DNA 16μl Digestion buffer 2μl Enzyme 1 (X) 1μl Enzyme 2 (P) 1μl run gel to check >> all failed. purification of (1)pLac+aptamer(XP) (2)terminator(XP) (3)pSB1C3(XP) ligation of (1)eIF4A+pSB1C3 (2)pLac+aptamer(SP)+terminator(XP) (3)J13002(SP)+GA/GB/RA/RB ligation protocol of GA,GB Total 10μl buffer 1μl ligase 0.5μl DNA1(pSB1A2) 2μl DNA2(GA/GB)) 5μl ddH2O 1.5μl ligation protocol of RA,RB Total 10μl buffer 1μl ligase 0.5μl DNA1(pSB1A2) 2μl DNA2(GA/GB)) 5μl ddH2O 1.5μl 2010.10.10 colony PCR of pSB1C3+eIF4A,pSB1C3+pLac+aptamer,J13002+ReB,J13002+GeB colony PCR protocol Total: 50μl X2.5μl Template 1μl FP(VF2) 1μl 2.5μl RP(VR) 1μl 2.5μl dNTP 2μl 5μl 10XBuff. 5μl 12.5μl tag 0.25μl 0.625μl ddH2O 39.75μl 99.375μl PCR Protocol 94 60s 94 15s 55 20s 72 100s 30 cycles 72 300s digestion of J13002 digestion protocol Total 20μl DNA 8μl Digestion buffer 2μl Enzyme 1 (S) 1μl Enzyme 2 (P) 1μl ddH2O 8μl ligation of pSB1C3+eIF4A ligaton protocol Total 10μl buffer 1μl ligase 0.5μl DNA1(pSB1C3) 2μl DNA2(eIF4A) 3μl ddH2O 3.5μl ligation of pSB1C3+pLac+aptamer,J13002+GeA,J13002+GeB ligation protocol Total 10μl buffer 1μl ligase 0.5μl DNA1(pSB1C3/J13002) 1.5μl DNA2(pLac+aptamer/GeA,GeB) 3.5μl ddH2O 3.5μl ligation ofJ13002+ReA,J13002+ReB ligation protocol Total 10μl buffer 1μl ligase 0.5μl DNA1(J13002) 2μl DNA2(ReA/ReB) 3μl ddH2O 3.5μl 2010.10.11 3in1 of pSB1C3+GeA,pSB1C3+GeB,pSB1C3+ReA,pSB1C3+ReB,pSB1C3+pLac+aptamer colony PCR protocol Total: 50μl X10μl Template 1μl FP 1μl RP 1μl dNTP 2μl 20μl 10XBuff. 5μl 50μl tag 0.25μl 2.5μl ddH2O 39.75μl 397.5μl PCR Protocol 94 60s 94 15s 55 20s 72 100s 30 cycles 72 300s 2010.10.12 digstion of J13002+GeA,J13002+ReA(x2),J13002+ReB(x2),pSB1C3+pLac+aptamer(x2) digestion protocol Total 20μl DNA 8μl Digestion buffer 2μl Enzyme 1 (S) 1μl Enzyme 2 (P) 1μl ddH2O 8μl 2010.10.13 digestion of J13002+GeA,J13002+ReA(X,P),J13002+ReB(x2),pSB1C3+pLac(S,P)→check length digestion protocol Total 20μl DNA 16μl Digestion buffer 2μl Enzyme 1 (X) 1μl Enzyme 2 (P) 1μl 3in1 of C3+GA(X1),C3+GB(X3), C3+eIF4A(X5) colony PCR protocol Total: 50μl X6μl Template 1μl FP 1μl RP 1μl dNTP 2μl 12μl 10XBuff. 5μl 30μl tag 0.25μl 1.5μl ddH2O 39.75μl 238.5μl PCR Protocol 94 60s 94 15s 55 20s 72 100s 30 cycles 72 300s run gel of JRA(X5),JRB(X2) ligation of (1)J13002+GA,GB,RA,GB (2)C3+GA,GB,RA,GB (3)C3+pLac+aptamer+terminator ligation protocol of GA,GB Total 10μl buffer 1μl ligase 0.5μl DNA1(pSB1A2) 2μl DNA2(GA/GB)) 5μl ddH2O 1.5μl 2010.10.14 3in1 of J13002+GA,GB,RA,RB (all failed) colony PCR protocol Total: 50μl X5μl Template 1μl FP 1μl RP 1μl dNTP 2μl 10μl 10XBuff. 5μl 25μl tag 0.25μl 1.25μl ddH2O 39.75μl 198.75μl PCR Protocol 94 60s 94 15s 55 20s 72 100s 30 cycles 72 300s real PCR of GeA,GeB,ReA,ReB Total: 50μl X5μl FP RP Template 1μl negative FP 1μl positive(eIF4A) eIF4A-Fp eIF4A-split-B-Rp RP 1μl GeA GFP-split-A-FP eIF4A-split-A-Rp dNTP 2μl 10μl GeB GFP-split-B-FP eIF4A-split-B-Rp 10XBuff. 5μl 25μl ReA RFP-split-A-FP eIF4A-split-A-Rp pfu 0.25μl 1.25μl ReB RFP-split-B-FP eIF4A-split-B-Rp ddH2O 39.75μl 198.75μl PCR Protocol 94 120s 94 30s 55 30s 72 100s 35 cycles 72 600s digestion of eIF4A(for biobrick) digestion protocol Total 20μl DNA 16μl Digestion buffer 2μl Enzyme 1 (X) 1μl Enzyme 2 (P) 1μl digestion of pLac+aptamer(SP) digestion protocol Total 20μl DNA 16μl Digestion buffer 2μl Enzyme 1 (S) 1μl Enzyme 2 (P) 1μl digestion of GeA,GeB,ReA,ReB,eIF4A(all from PCR, biobrick cutting site) digestion protocol Total 20μl DNA 16μl Digestion buffer 2μl Enzyme 1 (X) 1μl Enzyme 2 (P) 1μl tag PCR of negative > to check if there has contamination Total: 50μl X2μl FP RP Template 1μl negative FP 1μl positive(eIF4A) eIF4A-Fp eIF4A-split-B-Rp RP 1μl GeA GFP-split-A-FP eIF4A-split-A-Rp dNTP 2μl 4μl GeB GFP-split-B-FP eIF4A-split-B-Rp 10XBuff. 5μl 10μl ReA RFP-split-A-FP eIF4A-split-A-Rp tag 0.25μl 0.5μl ReB RFP-split-B-FP eIF4A-split-B-Rp ddH2O 39.75μl 79.5μl PCR Protocol 94 120s 94 30s 55 30s 72 100s 35 cycles 72 600s 2010.10.15 ligation of pSB1C3+GeA/GeB/ReA/ReB/eIF4A/pLac+eIF4A+treminator,pSB1A2+J13002+GeA/GeB/ReA/ReB ligation protocol ligation protocol of RA,RB Total 10μl buffer 1μl ligase 0.5μl DNA1() 4.5μl DNA2(~) 0.5μl ddH2O 3.5μl digestion of pSB1C3 digestion protocol Total 20μl DNA 16μl Digestion buffer 2μl Enzyme 1 (X) 1μl Enzyme 2 (P) 1μl PCR of GeA/ReA PCR protocol Total: 50μl X2μl FP RP Template 1μl negative FP 1μl positive(eIF4A) eIF4A-Fp eIF4A-split-B-Rp RP 1μl GeA GFP-split-A-FP eIF4A-split-A-Rp dNTP(KOD) 4μl 8μl GeB GFP-split-B-FP eIF4A-split-B-Rp 10XBuffer(KOD) 5μl 10μl ReA RFP-split-A-FP eIF4A-split-A-Rp KOD 1μl 2μl ddH2O 33μl 198.75μl MgSO4 4μl 8μl PCR Protocol 94 120s 94 30s 55 30s 72 100s 35 cycles 72 600s 3in1 of J13002+GB,pSB1C3+pLac+aptamer+terminator colony PCR protocol Total: 50μl X3μl Template 1μl FP 1μl 3μl RP 1μl 3μl dNTP 2μl 6μl 10XBuff. 5μl 15μl tag 0.25μl 0.75μl ddH2O 39.75μl 119.75μl PCR Protocol 94 60s 94 15s 55 20s 72 100s 30 cycles 72 300s 2010.10.16 ligation of pSB1C3+GeA/GeB/ReA/ReB/eIF4A/pLac+eIF4A+treminator,pSB1A2+J13002+GeA/GeB/ReA/ReB ligation protocol Total 10μl buffer 1μl ligase 0.5μl DNA1(J13002) 4.5μl DNA2(~) 0.5μl ddH2O 3.5μl digestion of C3+GeA(X,P)/GeB(X,P)/ReA(X,P),J13002+GeB(S,P) digestion protocol Total 20μl DNA 16μl Digestion buffer 2μl Enzyme 1 (X,S) 1μl Enzyme 2 (P) 1μl 2010.10.17 3in1 of J13002+ReA/GeA/ReB/GeB+terminator colony PCR protocol Total: 50μl X3.6μl Template 1μl FP 1μl 3.6μl RP 1μl 3.6μl dNTP 2μl 7.2μl 10XBuff. 5μl 18μl tag 0.25μl 0.9μl ddH2O 39.75μl 141.3μl PCR Protocol 94 60s 94 15s 55 20s 72 100s 30 cycles 72 300s ligation of pSB1C3+GeB/eIF4A,pSB1A2+J13002+GeB ligation protocol ligation protocol of RA,RB Total 10μl buffer 1μl ligase 0.5μl DNA1(J13002) 1μl DNA2(~) 2μl ddH2O 5.5μl 2010.10.18 3in1 of JGeA, JReA, JReB, C3+ReB colony PCR protocol Total: 50μl X6μl Template 1μl FP 1μl RP 1μl dNTP 2μl 12μl 10XBuff. 5μl 30μl tag 0.25μl 1.5μl ddH2O 39.75μl 238.5μl PCR Protocol 94 60s 94 15s 55 20s 72 100s 30 cycles 72 300s 2010.10.19 ligation of JReA, JReB, C3+GeA,GeB,ReA,eIF4A ligation protocol Total 10μl buffer 1μl ligase 0.5μl DNA1(vector) 1μl DNA2(~)) 2μl ddH2O 5.5μl digestion of JGeA digestion protocol Total 20μl DNA 10μl Digestion buffer 2μl Enzyme 1 (S) 1μl Enzyme 2 (P) 1μl ddH2O 6μl 2010.10.20 3in1 of (1)JGeA+terminator (2)J13002+ReA(old) (3)J13002+ReA(new) colony PCR protocol Total: 50μl X2μl Template 1μl FP 1μl RP 1μl dNTP 2μl 4μl 10XBuff. 5μl 10μl tag 0.25μl 0.5μl ddH2O 39.75μl 79.5μl PCR Protocol 94 60s 94 15s 55 20s 72 100s 30 cycles 72 300s ligation of (1)JGeA+terminator (2)JReA (3)JReB (4)C3+GeA (5)C3+GeB (6)C3+ReA (7)C3+ReB (8)C3+eIF4A ligation protocol of GA,GB Total 10μl buffer 1μl ligase 0.5μl DNA1(vector) 1μl DNA2(~) 2μl ddH2O 5.5μl 2010.10.21 ligation of J13002 ReB,pSB1C3+ReA/ReB/GeA/GeB/eIF4A ligation protocol Total 10μl buffer 1μl ligase 0.5μl DNA1(vector) 1μl DNA2(~) 2μl ddH2O 5.5μl 2010.10.22 ligation of J13002+ReA/ReB,pSB1C3+eIF4A/GeA/GeB/ReA/ReB ligation protocol Total 10μl buffer 1μl ligase 0.5μl DNA1(vector) 1μl DNA2(~) 2μl ddH2O 5.5μl 2010.10.23 3in1 of JGeA, JReA, JReB colony PCR protocol Total: 50μl X5μl Template 1μl FP 1μl RP 1μl dNTP 2μl 10μl 10XBuff. 5μl 25μl tag 0.25μl 1.25μl ddH2O 39.75μl 198.75μl PCR Protocol 94 60s 94 15s 55 20s 72 100s 30 cycles 72 300s 2010.10.24 3in1 of JReA, JReB, C3+ReB/eIF4A/GA/GB/RA colony PCR protocol Total: 50μl X6.6μl Template 1μl FP 1μl RP 1μl dNTP 2μl 13.2μl 10XBuff. 5μl 33μl tag 0.25μl 1.65μl ddH2O 39.75μl 262.35μl PCR Protocol 94 60s 94 15s 55 20s 72 100s 30 cycles 72 300s digestion of JGeAT,JGeT digestion protocol Total 20μl DNA 16μl Digestion buffer 2μl Enzyme 1 (X) 1μl Enzyme 2 (P) 1μl 2010.10.25 2010.10.26 ligation of pSB1C3+eIF4A/GeA/ReA/ReB ligation protocol Total 10μl buffer 1μl ligase 0.5μl DNA1(vector) 1μl DNA2(~) 2μl ddH2O 5.5μl 3in1 of pSB1C3+GeA/ReA/ReB 2010.10.27 2010.10.28 NYMU-Taipei iGEM 2010 Team