537-psPAX2-D64V
Cloning of 537-psPAX2-D64V
[edit]Purpose: Cloning of 537-psPAX2-D64V by subclone from 514 pLV-Gag-PolD64V to 503 psPAX2
Date: 2012-1-24--2012-1-26
Notebook:
Page:
Clone ID:537
Experimenters: yingying
Results
[edit]
Subcloning
[edit]Date: 2012-1-24
| band 1 |
band 2 | |
| 514 pLV-Gag-PolD64V |
503 psPAX2 | |
| digestion enzymes |
Swa1 | |
Colony Screening
[edit]
Result intepretation/Summary
[edit]Conclusion
[edit]
Cloning of 537-psPAX2-D64V
[edit]Purpose: Cloning of 537-psPAX2-D64V by subclone from 514 pLV-Gag-PolD64V to 503 psPAX2
Date: 2012-4-5--2012-4-6
Notebook:
Page:
Clone ID:537
Experimenters: yingying
Results
[edit]
Subcloning
[edit]Date: 2012-4-4
| band 1 |
band 2 | |
| 514 pLV-Gag-PolD64V |
503 psPAX2 | |
| digestion enzymes |
Swa1+AflII | |
first digest with Swa1 for 4 hours at 25c,then digest with AflII for 4 hours at 37c, then run gel ,can not cut out mutated Pol gene fragment from 514 pLV-Gag-PolD64V using Swa1 + AflI
Colony Screening
[edit]
Result intepretation/Summary
[edit]Conclusion
[edit]
Cloning of 537-psPAX2-D64V
[edit]Purpose: Cloning of 537-psPAX2-D64V
Date: 2012-4-16---2012-4-18
Notebook:0
Page:
Clone ID:537
Experimenters: yingying
Results
[edit]subcloning
[edit]
first digest with Swa1 for 4 hours at 25c
2nd digest with AflII for 4 hours at 37c
| band 1 |
band 2 | |
| 514 pLV-Gag-PolD64V |
503 psPAX2 | |
| enzymes |
Swa1+AflII. | |
Colony Screening
[edit]Date: 2012-4-18
Make miniprep and sequencing
no right clone
Result intepretation/Summary
[edit]- <br>
Conclusion
[edit]<br>
<br>
<br>
Cloning of 537-psPAX2-D64V
[edit]Purpose: Cloning of 537-psPAX2-D64V
Date: 2012-5-2---2012-5-4
Notebook:0
Page:
Clone ID:537
Experimenters: yingying
Results
[edit]cloning
[edit]
PCR:
PCR1:Int-D64V-F CCAGGAATATGGCAGCTAGTTTG TACACATTTAGAAGG + PgagPol-Seq-R CTTAAAATTAGCAGGAAGATGG
PCR2:pGagPol-Seq-F AGTGATTTTAACCTACCACC +Int-D64V-R CCTTCTAAATGTGTACAAAC TAGCTGCCATATTCCTGG
PCR1 NO BAND, because of the wrong primer
Colony Screening
[edit]
Result intepretation/Summary
[edit]- <br>
Conclusion
[edit]<br>
<br>
<br>
Cloning of 537-psPAX2-D64V
[edit]Purpose: Cloning of 537-psPAX2-D64V by subclone from 514 pLV-Gag-PolD64V to 503 psPAX2
Date: 2012-5-9--2012-5-13
Notebook:
Page:
Clone ID:537
Experimenters: yingying
Results
[edit]
Subcloning
[edit]Date: 2012-5-9
PCR1:Int-D64V-F CCAGGAATATGGCAGCTAGTTTG TACACATTTAGAAGG +PgagPol-Seq-R CTTAAAATTAGCAGGAAGATGG
PgagPol-Seq-R2 ccatcttcctgctaattttaag
PCR2:pGagPol-Seq-F AGTGATTTTAACCTACCACC +Int-D64V-R CCTTCTAAATGTGTACAAAC TAGCTGCCATATTCCTGG
PCR:PCR1+PCR2 as template
PgagPol-Seq-R2 ccatcttcctgctaattttaag+pGagPol-Seq-F AGTGATTTTAACCTACCACC
| band 1 |
band 2 | |
| digestion enzymes |
| |
5/14/2012
514 digestion with different enzymes
| Lane | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| Enzyme | Nde1 | EcoR1 | Nde1+ EcoR1 | Nde1+Sal1 | Nde1+BamH1 | Nde1+ Xho1 | Nde1+AflII |
| Fragments | 2 | 2 | 4 | 2 | 3 | 2 | 2 |
Colony Screening
[edit]
Result intepretation/Summary
[edit]Conclusion
[edit]
2012-5-18----2012-5-
[edit]
| band 1 |
band 2 | |
| 514 pLV-Gag-PolD64V |
503 psPAX2 | |
| Enzymes |
EcoR1 | |
PCR screen:2012-5-22
2012-5-23
pick 6 clonies for digesttion detect: EcoR1
no insert
2012-6-22-----2012-6-28
| lane |
1 |
2 |
| 514 pLV-Gag-PolD64V |
503 psPAX2 | |
| enzymes |
EcoR1 | |
2012-6-26
digest detect:
all have insert
2012-6-28
Cloning of 537-psPAX2-D64V
[edit]Purpose: Cloning of 537-psPAX2-D64V
Date: 2012-6-28---2012-7-20
Notebook:0
Page:
Clone ID:537
Experimenters: yingying
Results
[edit]subcloning
[edit]
| band 1 |
band 2 | |
| 503 psPAX2 |
514 pLV-Gag-PolD64V | |
| enzymes |
EcoRV+AflII | |
Colony Screening
[edit]Date: 2012-7-5
Make miniprep and digest with EcoR1
Clone 2,4,5,6 may be right, PCR test
Int-D64V-F CCAGGAATATGGCAGCTAGTTTG TACACATTTAGAAGG + PgagPol-Seq-R2 ccatcttcctgctaattttaag
clone2,4,5,6 right
2012-7-16-----2012-7-20
pick 8 clones and do miniprep again
digest with EcoR1
Clone 2,3,4,5,7,8 may be right
2012-7-20
PCR screen using pGagPol-Seq-F+pGagPol-Seq-R2
choose clone3 PCR product for sequencing
Result intepretation/Summary
[edit]- <br>
Conclusion
[edit]<br>
<br>
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