MRNA Binding Experiments
From 2010.igem.org
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(→2010.10.24) |
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- | [[Image:NYMU_CIMG5632.JPG|left|400px|thumb| | + | [[Image:NYMU_CIMG5632.JPG|left|400px|thumb|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.]] |
{| | {| | ||
|- | |- | ||
| | | | ||
- | |||
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|} | |} | ||
*run gel and gel extraction | *run gel and gel extraction | ||
- | [[Image:NYMU_CIMG5633.JPG|left|400px|thumb| | + | [[Image:NYMU_CIMG5633.JPG|left|400px|thumb|band1~10 → apt.+ter.(XP) // maker // eIF4A ]] |
{| | {| | ||
|- | |- | ||
Line 724: | Line 723: | ||
*ligation of apt.+ter(XP)+(RBS(SP) | *ligation of apt.+ter(XP)+(RBS(SP) | ||
*transformation | *transformation | ||
+ | |||
=2010.08.31= | =2010.08.31= | ||
*3 in 1 of '''apt+term+RBS''' | *3 in 1 of '''apt+term+RBS''' | ||
Line 847: | Line 847: | ||
|- | |- | ||
|} | |} | ||
- | [[Image:NYMU_CIMG5635.JPG|left|400px|thumb|mut.2 and3 didn't have | + | [[Image:NYMU_CIMG5635.JPG|left|400px|thumb|mut.2 and3 didn't have bands, so we PCR test again. ]] |
{| | {| | ||
|- | |- | ||
Line 1,316: | Line 1,316: | ||
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+ | [[image:NYMU_2010_10_28.png|left|400px|thumb| ]] | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
+ | |||
=2010.09.10= | =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><) | *3 in 1 of pSB1C3+pLac+RBS+apt+term and pSB1C3+RBS+apt+term(because LB tube did not be put for cultivating><) | ||
Line 1,380: | Line 1,385: | ||
=2010.09.11= | =2010.09.11= | ||
*plasmid extraction of pSB1C3+pLac+RBS+aptamer+terminator and pSB1C3+RBS+aptamer+terminator succeed! Have two new 2010 biobricks! | *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 | + | *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. | *The plate of pSB1C3+aptamer+terminator have no colonies, so ligate again. | ||
*digestion of pSB1C3(XP) | *digestion of pSB1C3(XP) | ||
Line 1,416: | Line 1,421: | ||
|- | |- | ||
|} | |} | ||
+ | [[image:NYMU_2010_9_11.png|left|400px|thumb| ]] | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
=2010.09.12= | =2010.09.12= | ||
Line 1,561: | Line 1,570: | ||
|- | |- | ||
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- | + | [[image:NYMU_2010_9_14.png|left|400px|thumb| ]] | |
+ | {| | ||
+ | |- | ||
+ | | | ||
=2010.9.15= | =2010.9.15= | ||
Line 1,757: | Line 1,769: | ||
|- | |- | ||
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+ | [[image:NYMU_2010_9_16.png|left|400px|thumb| ]] | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
=2010.9.17= | =2010.9.17= | ||
Line 1,970: | Line 1,986: | ||
|- | |- | ||
|} | |} | ||
+ | [[image:NYMU_2010_9_21.png|left|400px|thumb| ]] | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
+ | [[image:NYMU_2010_9_21_1.png|left|400px|thumb| ]] | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
=2010.9.22= | =2010.9.22= | ||
Line 2,027: | Line 2,051: | ||
|- | |- | ||
|} | |} | ||
+ | [[image:NYMU_2010_9_22.png|left|400px|thumb| ]] | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
+ | [[image:NYMU_2010_9_22_1.png|left|400px|thumb| ]] | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
=2010.9.23= | =2010.9.23= | ||
Line 2,112: | Line 2,144: | ||
|- | |- | ||
|} | |} | ||
+ | [[image:NYMU_2010_9_24.png|left|400px|thumb| ]] | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
+ | [[image:NYMU_2010_9_24_1.png|left|400px|thumb| ]] | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
+ | |||
=2010.09.25= | =2010.09.25= | ||
*plasmid extration of | *plasmid extration of | ||
Line 2,196: | Line 2,237: | ||
|- | |- | ||
|} | |} | ||
+ | [[image:NYMU_2010_9_25.png|left|400px|thumb| ]] | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
+ | [[image:NYMU_2010_9_25_1.png|left|400px|thumb| ]] | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
+ | |||
=2010.09.26= | =2010.09.26= | ||
*colony PCR of GA(X2),GB(X2),RA(X2),RB(X2),on pSB1A2 | *colony PCR of GA(X2),GB(X2),RA(X2),RB(X2),on pSB1A2 | ||
Line 2,243: | Line 2,293: | ||
|} | |} | ||
*digestion and gel extration of pSB1C3(XP), pSB1A2(XP) | *digestion and gel extration of pSB1C3(XP), pSB1A2(XP) | ||
+ | [[image:NYMU_2010_9_26.png|left|400px|thumb| ]] | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
+ | |||
=2010.09.27= | =2010.09.27= | ||
*plasmid extraction of GFP-e-A-2,GFP-e-B-2,RFP-e-A-2,RFP-e-B-2 | *plasmid extraction of GFP-e-A-2,GFP-e-B-2,RFP-e-A-2,RFP-e-B-2 | ||
Line 2,298: | Line 2,353: | ||
|} | |} | ||
*transformation. | *transformation. | ||
+ | [[image:NYMU_2010_9_27.png|left|400px|thumb| ]] | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
+ | [[image:NYMU_2010_9_27_1.png|left|400px|thumb| ]] | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
+ | [[image:NYMU_2010_9_27_2.png|left|400px|thumb| ]] | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
+ | |||
=2010.09.28= | =2010.09.28= | ||
*3 in 1 of colony PCR of GA,GB,RA,RB | *3 in 1 of colony PCR of GA,GB,RA,RB | ||
Line 2,344: | Line 2,412: | ||
|- | |- | ||
|} | |} | ||
+ | [[image:NYMU_2010_9_28.png|left|400pixel|thumb| ]] | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
+ | |||
=2010.09.29= | =2010.09.29= | ||
*plasmid extractio of GA(1),GB(2),RA(1),RB(2) | *plasmid extractio of GA(1),GB(2),RA(1),RB(2) | ||
Line 2,506: | Line 2,579: | ||
|- | |- | ||
|} | |} | ||
+ | [[image:NYMU_2010_10_01.png|left|400px|thumb| ]] | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
=2010.10.02= | =2010.10.02= | ||
Line 3,241: | Line 3,318: | ||
|- | |- | ||
|} | |} | ||
- | + | *real PCR of GeA,GeB,ReA,ReB | |
+ | {| border=1 | ||
+ | |- | ||
+ | ! 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 | ||
+ | |} | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
+ | {| border="1" | ||
+ | |- | ||
+ | ! PCR Protocol !! | ||
+ | |- | ||
+ | | 94 | ||
+ | | 120s | ||
+ | |- | ||
+ | | 94 | ||
+ | | 30s | ||
+ | |- | ||
+ | | 55 | ||
+ | | 30s | ||
+ | |- | ||
+ | | 72 | ||
+ | | 100s | ||
+ | | 35 cycles | ||
+ | |- | ||
+ | | 72 | ||
+ | | 600s | ||
+ | |- | ||
+ | |} | ||
+ | *digestion of eIF4A(for biobrick) | ||
+ | **digestion protocol | ||
+ | |||
+ | {| border=1 | ||
+ | |- | ||
+ | ! Total !! 20μl | ||
+ | |- | ||
+ | | DNA || 16μl | ||
+ | |- | ||
+ | | Digestion buffer || 2μl | ||
+ | |- | ||
+ | | Enzyme 1 (X) || 1μl | ||
+ | |- | ||
+ | | Enzyme 2 (P) || 1μl | ||
+ | |- | ||
+ | |} | ||
+ | *digestion of pLac+aptamer(SP) | ||
+ | **digestion protocol | ||
+ | |||
+ | {| border=1 | ||
+ | |- | ||
+ | ! 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 | ||
+ | |||
+ | {| border=1 | ||
+ | |- | ||
+ | ! 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 | ||
+ | {| border=1 | ||
+ | |- | ||
+ | ! 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 | ||
+ | |} | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
+ | {| border="1" | ||
+ | |- | ||
+ | ! PCR Protocol !! | ||
+ | |- | ||
+ | | 94 | ||
+ | | 120s | ||
+ | |- | ||
+ | | 94 | ||
+ | | 30s | ||
+ | |- | ||
+ | | 55 | ||
+ | | 30s | ||
+ | |- | ||
+ | | 72 | ||
+ | | 100s | ||
+ | | 35 cycles | ||
+ | |- | ||
+ | | 72 | ||
+ | | 600s | ||
+ | |- | ||
+ | |} | ||
=2010.10.15= | =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 | ||
+ | |||
+ | {| border=1 | ||
+ | |- | ||
+ | ! Total !! 10μl | ||
+ | |- | ||
+ | | buffer || 1μl | ||
+ | |- | ||
+ | | ligase || 0.5μl | ||
+ | |- | ||
+ | | DNA1() || 4.5μl | ||
+ | |- | ||
+ | | DNA2(~) || 0.5μl | ||
+ | |- | ||
+ | | ddH2O || 3.5μl | ||
+ | |- | ||
+ | |} | ||
+ | |||
+ | *digestion of pSB1C3 | ||
+ | **digestion protocol | ||
+ | {| border=1 | ||
+ | |- | ||
+ | ! Total !! 20μl | ||
+ | |- | ||
+ | | DNA || 16μl | ||
+ | |- | ||
+ | | Digestion buffer || 2μl | ||
+ | |- | ||
+ | | Enzyme 1 (X) || 1μl | ||
+ | |- | ||
+ | | Enzyme 2 (P) || 1μl | ||
+ | |- | ||
+ | |} | ||
+ | |||
+ | *PCR of GeA/ReA | ||
+ | **PCR protocol | ||
+ | {| border=1 | ||
+ | |- | ||
+ | ! 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 | ||
+ | |} | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
+ | {| border="1" | ||
+ | |- | ||
+ | ! 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 | ||
+ | |||
+ | {| border=1 | ||
+ | |- | ||
+ | ! 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 | ||
+ | |} | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
+ | {| border="1" | ||
+ | |- | ||
+ | ! PCR Protocol !! | ||
+ | |- | ||
+ | | 94 | ||
+ | | 60s | ||
+ | |- | ||
+ | | 94 | ||
+ | | 15s | ||
+ | |- | ||
+ | | 55 | ||
+ | | 20s | ||
+ | |- | ||
+ | | 72 | ||
+ | | 100s | ||
+ | | 30 cycles | ||
+ | |- | ||
+ | | 72 | ||
+ | | 300s | ||
+ | |- | ||
+ | |} | ||
+ | |||
=2010.10.16= | =2010.10.16= | ||
+ | *ligation of pSB1C3+GeA/GeB/ReA/ReB/eIF4A/pLac+eIF4A+treminator,pSB1A2+J13002+GeA/GeB/ReA/ReB | ||
+ | **ligation protocol | ||
+ | |||
+ | {| border=1 | ||
+ | |- | ||
+ | ! 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 | ||
+ | {| border=1 | ||
+ | |- | ||
+ | ! Total !! 20μl | ||
+ | |- | ||
+ | | DNA || 16μl | ||
+ | |- | ||
+ | | Digestion buffer || 2μl | ||
+ | |- | ||
+ | | Enzyme 1 (X,S) || 1μl | ||
+ | |- | ||
+ | | Enzyme 2 (P) || 1μl | ||
+ | |- | ||
+ | |} | ||
+ | |||
=2010.10.17= | =2010.10.17= | ||
+ | *3in1 of J13002+ReA/GeA/ReB/GeB+terminator | ||
+ | **colony PCR protocol | ||
+ | |||
+ | {| border=1 | ||
+ | |- | ||
+ | ! 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 | ||
+ | |} | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
+ | {| border="1" | ||
+ | |- | ||
+ | ! 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 | ||
+ | |||
+ | {| border=1 | ||
+ | |- | ||
+ | ! Total !! 10μl | ||
+ | |- | ||
+ | | buffer || 1μl | ||
+ | |- | ||
+ | | ligase || 0.5μl | ||
+ | |- | ||
+ | | DNA1(J13002) || 1μl | ||
+ | |- | ||
+ | | DNA2(~) || 2μl | ||
+ | |- | ||
+ | | ddH2O || 5.5μl | ||
+ | |- | ||
+ | |} | ||
+ | |||
=2010.10.18= | =2010.10.18= | ||
+ | *3in1 of JGeA, JReA, JReB, C3+ReB | ||
+ | **colony PCR protocol | ||
+ | |||
+ | {| border=1 | ||
+ | |- | ||
+ | ! 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 | ||
+ | |} | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
+ | {| border="1" | ||
+ | |- | ||
+ | ! PCR Protocol !! | ||
+ | |- | ||
+ | | 94 | ||
+ | | 60s | ||
+ | |- | ||
+ | | 94 | ||
+ | | 15s | ||
+ | |- | ||
+ | | 55 | ||
+ | | 20s | ||
+ | |- | ||
+ | | 72 | ||
+ | | 100s | ||
+ | | 30 cycles | ||
+ | |- | ||
+ | | 72 | ||
+ | | 300s | ||
+ | |- | ||
+ | |} | ||
+ | |||
=2010.10.19= | =2010.10.19= | ||
+ | *ligation of JReA, JReB, C3+GeA,GeB,ReA,eIF4A | ||
+ | **ligation protocol | ||
+ | |||
+ | {| border=1 | ||
+ | |- | ||
+ | ! Total !! 10μl | ||
+ | |- | ||
+ | | buffer || 1μl | ||
+ | |- | ||
+ | | ligase || 0.5μl | ||
+ | |- | ||
+ | | DNA1(vector) || 1μl | ||
+ | |- | ||
+ | | DNA2(~)) || 2μl | ||
+ | |- | ||
+ | | ddH2O || 5.5μl | ||
+ | |- | ||
+ | |} | ||
+ | *digestion of JGeA | ||
+ | **digestion protocol | ||
+ | |||
+ | {| border=1 | ||
+ | |- | ||
+ | ! Total !! 20μl | ||
+ | |- | ||
+ | | DNA || 10μl | ||
+ | |- | ||
+ | | Digestion buffer || 2μl | ||
+ | |- | ||
+ | | Enzyme 1 (S) || 1μl | ||
+ | |- | ||
+ | | Enzyme 2 (P) || 1μl | ||
+ | |- | ||
+ | | ddH2O || 6μl | ||
+ | |- | ||
+ | |} | ||
+ | |||
=2010.10.20= | =2010.10.20= | ||
+ | *3in1 of | ||
+ | (1)JGeA+terminator | ||
+ | |||
+ | (2)J13002+ReA(old) | ||
+ | |||
+ | (3)J13002+ReA(new) | ||
+ | **colony PCR protocol | ||
+ | |||
+ | {| border=1 | ||
+ | |- | ||
+ | ! 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 | ||
+ | |} | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
+ | {| border="1" | ||
+ | |- | ||
+ | ! 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 | ||
+ | |||
+ | {| border=1 | ||
+ | |- | ||
+ | ! Total !! 10μl | ||
+ | |- | ||
+ | | buffer || 1μl | ||
+ | |- | ||
+ | | ligase || 0.5μl | ||
+ | |- | ||
+ | | DNA1(vector) || 1μl | ||
+ | |- | ||
+ | | DNA2(~) || 2μl | ||
+ | |- | ||
+ | | ddH2O || 5.5μl | ||
+ | |- | ||
+ | |} | ||
+ | |||
=2010.10.21= | =2010.10.21= | ||
+ | *ligation of J13002 ReB,pSB1C3+ReA/ReB/GeA/GeB/eIF4A | ||
+ | **ligation protocol | ||
+ | |||
+ | {| border=1 | ||
+ | |- | ||
+ | ! Total !! 10μl | ||
+ | |- | ||
+ | | buffer || 1μl | ||
+ | |- | ||
+ | | ligase || 0.5μl | ||
+ | |- | ||
+ | | DNA1(vector) || 1μl | ||
+ | |- | ||
+ | | DNA2(~) || 2μl | ||
+ | |- | ||
+ | | ddH2O || 5.5μl | ||
+ | |- | ||
+ | |} | ||
+ | |||
=2010.10.22= | =2010.10.22= | ||
+ | *ligation of J13002+ReA/ReB,pSB1C3+eIF4A/GeA/GeB/ReA/ReB | ||
+ | **ligation protocol | ||
+ | |||
+ | {| border=1 | ||
+ | |- | ||
+ | ! Total !! 10μl | ||
+ | |- | ||
+ | | buffer || 1μl | ||
+ | |- | ||
+ | | ligase || 0.5μl | ||
+ | |- | ||
+ | | DNA1(vector) || 1μl | ||
+ | |- | ||
+ | | DNA2(~) || 2μl | ||
+ | |- | ||
+ | | ddH2O || 5.5μl | ||
+ | |- | ||
+ | |} | ||
+ | |||
=2010.10.23= | =2010.10.23= | ||
+ | *3in1 of JGeA, JReA, JReB | ||
+ | **colony PCR protocol | ||
+ | |||
+ | {| border=1 | ||
+ | |- | ||
+ | ! 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 | ||
+ | |} | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
+ | {| border="1" | ||
+ | |- | ||
+ | ! PCR Protocol !! | ||
+ | |- | ||
+ | | 94 | ||
+ | | 60s | ||
+ | |- | ||
+ | | 94 | ||
+ | | 15s | ||
+ | |- | ||
+ | | 55 | ||
+ | | 20s | ||
+ | |- | ||
+ | | 72 | ||
+ | | 100s | ||
+ | | 30 cycles | ||
+ | |- | ||
+ | | 72 | ||
+ | | 300s | ||
+ | |- | ||
+ | |} | ||
+ | |||
=2010.10.24= | =2010.10.24= | ||
+ | *3in1 of JReA, JReB, C3+ReB/eIF4A/GA/GB/RA | ||
+ | **colony PCR protocol | ||
+ | |||
+ | {| border=1 | ||
+ | |- | ||
+ | ! 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 | ||
+ | |} | ||
+ | {| | ||
+ | |- | ||
+ | | | ||
+ | {| border="1" | ||
+ | |- | ||
+ | ! PCR Protocol !! | ||
+ | |- | ||
+ | | 94 | ||
+ | | 60s | ||
+ | |- | ||
+ | | 94 | ||
+ | | 15s | ||
+ | |- | ||
+ | | 55 | ||
+ | | 20s | ||
+ | |- | ||
+ | | 72 | ||
+ | | 100s | ||
+ | | 30 cycles | ||
+ | |- | ||
+ | | 72 | ||
+ | | 300s | ||
+ | |- | ||
+ | |} | ||
+ | [[image:DSC05773.JPG|left|400px|thumb| ]] | ||
+ | *digestion of JGeAT,JGeT | ||
+ | **digestion protocol | ||
+ | {| border=1 | ||
+ | |- | ||
+ | ! Total !! 20μl | ||
+ | |- | ||
+ | | DNA || 16μl | ||
+ | |- | ||
+ | | Digestion buffer || 2μl | ||
+ | |- | ||
+ | | Enzyme 1 (X) || 1μl | ||
+ | |- | ||
+ | | Enzyme 2 (P) || 1μl | ||
+ | |- | ||
+ | |} | ||
+ | |||
=2010.10.25= | =2010.10.25= | ||
=2010.10.26= | =2010.10.26= | ||
- | = | + | *ligation of pSB1C3+eIF4A/GeA/ReA/ReB |
- | + | **ligation protocol | |
+ | |||
+ | {| border=1 | ||
+ | |- | ||
+ | ! Total !! 10μl | ||
+ | |- | ||
+ | | buffer || 1μl | ||
+ | |- | ||
+ | | ligase || 0.5μl | ||
+ | |- | ||
+ | | DNA1(vector) || 1μl | ||
+ | |- | ||
+ | | DNA2(~) || 2μl | ||
+ | |- | ||
+ | | ddH2O || 5.5μl | ||
+ | |- | ||
+ | |} | ||
+ | |||
+ | *3in1 of pSB1C3+GeA/ReA/ReB | ||
+ | |||
{{:Team:NYMU-Taipei/Footer}} | {{:Team:NYMU-Taipei/Footer}} |
Latest revision as of 03:53, 28 October 2010
Home | Project Overview | Speedy reporter | Speedy switch | Speedy protein degrader | Experiments and Parts | Applications | F.A.Q | About Us |
2010.08.09
- PCR of split GFP(A.B)
- PCR of split GFP (test/+tag/5 tubes)
Total: | 50μl | X2.8μl | FP | RP | Template | ||
---|---|---|---|---|---|---|---|
Template | 1μl | 5.6μl | negative | VF2 | VR | ||
FP | 1μl | positive | VF2 | VR | complete GFP | ||
RP | 1μl | GFP_A+B | GFP_split_A_FP | GFP_split_B_RP | complete GFP | ||
dNTP | 2μl | 5.6μl | GFP_A | GFP_split_A_FP | GFP_split_A_RP | complete GFP | |
10XBuff. | 5μl | 14μl | GFP_B | GFP_split_B_FP | GFP_split_B_RP | complete GFP | |
taq | 0.25μl | 0.7μl | |||||
ddH2O | 39.75μl | 111.3μl |
|