493-pLVXPlus2-App-IRES-mCherry: Difference between revisions
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Latest revision as of 15:28, 6 January 2012
Cloning of 493-pLVXPlus2-App-IRES-mCherry
Purpose: to subclone from 303 pLentiGP-App to pLVXPlus2-IRES-mCherry, 174
Date: 2011-11-10----
Notebook:Lu yingying
Page:
Clone ID:493
Experimenters: lu yingying
Results
method1:
digest 303 with BamH1 + Nhe1--> insert(band3), ligate to BamH1 + PmeI digested pLVXPlus2-IRES-mCherry, 174(band4).
purify with OMEGA column then add pfu polymerase + dNTP to fill in (37C,1hour), ligate again.
transform STBL3.
digest detect: BamH1+Xho1
Date: 2011-11-15----
Notebook:Lu yingying
Page:
Clone ID:493
Experimenters: lu yingying
Results
method1:
digest 303 with BamH1 + Nhe1--> insert(band3), ligate to BamH1 + PmeI digested pLVXPlus2-IRES-mCherry, 174(band4).
purify with OMEGA column then add pfu polymerase + dNTP to fill in (37C,1hour), ligate again.
transform STBL3.
Date: 2011-11-23----
Notebook:Lu yingying
Page:
Clone ID:493
Experimenters: lu yingying
Results
method2:
PCR template is pCMV-SPORT6-App 305
using these primers
App-BamH1-F
atcga ggatcc gcc acc atgctgcccagcctggcactg
App-HindIII-Sfi1-R
(gttctt tgagcagatg cagaactaa AAGCTT GGCCAAACAGGC atcgact)
agtcgatGCCTGTTTGGCCAAGCTTttagttctgcatctgctcaaagaac
band 1,2,3 use high GC buffer because of the GC content,
band 4,5,6 use HIFi buffer
Digest PCR product with BamH1 + Sfi1, digest pLVXPlus2-IRES-mCherry(174) vector with BamH1 + Sfi1
Date: 2011-11-28----
Notebook:Lu yingying
Page:
Clone ID:493
Experimenters: lu yingying
Results
Digest PCR product (band 2) with BamH1 + Sfi1, digest pLVXPlus2-IRES-mCherry(174) vector with BamH1 + Sfi1(band 3)
digest detect:BamH1 + Sfi1
Date: 2011-12-2----
Notebook:Lu yingying
Page:
Clone ID:493
Experimenters: lu yingying
Results
1st Digest PCR product (band 1) with BamH1 , digest pLVXPlus2-IRES-mCherry(174) vector with BamH1 (band2)
2nd Digest PCR product (band 1) with Sfi1 , digest pLVXPlus2-IRES-mCherry(174) vector with Sfi1 (band2)
1st digest (band 1) with BamH1 ,then digest with Sfi1
Date: 2011-12-12----
Notebook:Lu yingying
Page:
Clone ID:493
Experimenters: lu yingying
Results
BamH1 NEB , for 2 hours ,then Sfil1 ,NEB, for 5 hours in waterbath
BamH1 NEB ,for 2 hours ,then Sfil1 ,NEB, for 2 hours in waterbath
--Yingying.lu 10:06, 16 December 2011 (EST)
Cloning of 493-pLVXPlus2-App-IRES-mCherry
[edit]Purpose: Cloning of 493-pLVXPlus2-App-IRES-mCherry by PCR
Date: 2011-12-21---
Notebook:
Page:
Clone ID:493
Experimenters: yingying
Results
[edit]
method2:
PCR template is pCMV-SPORT6-App 305
using these primers
App-BamH1-F
atcga ggatcc gcc acc atgctgcccagcctggcactg
App-HindIII-Sfi1-R
(gttctt tgagcagatg cagaactaa AAGCTT GGCCAAACAGGC atcgact)
agtcgatGCCTGTTTGGCCAAGCTTttagttctgcatctgctcaaagaac
use HIFi buffer
BamH1 NEB ,for 2 hours ,then Sfil1 ,NEB, for 4 hours in waterbath
| band 5 |
band 6 | |
| PCR product (smear) |
pLVXPlus2-IRES-mCherry(174) | |
| digest |
BamH1 + Sfi1 |
BamH1 + Sfi1 |
BamH1 NEB ,for 2 hours ,then Sfil1 ,NEB, for 2 hours in waterbath
Subcloning
[edit]Date:
Colony Screening
[edit]Date: 2011-12-29
Make miniprep and digest with BamH1 + Sfi1
Result intepretation/Summary
[edit]Conclusion
[edit]
Cloning of 493-pLVXPlus2-App-IRES-mCherry
[edit]Purpose: Cloning of 493-pLVXPlus2-App-IRES-mCherry by PCR
Date: 2012-1-3
Notebook:
Page:
Clone ID:493
Experimenters: yingying
Results
[edit]
method3:
App-Pme1-R gctagagcGTTTAAACttagttctgcatctgctcaaag
App-BamH1-F atcga ggatcc gcc acc atgctgcccagcctggcactg.
Template pCMV-SPORT6-App 305, use HiFi buffer
| band 5 |
band 6 | |
| PCR product |
pLVXPlus2-IRES-mCherry(174) | |
| digest enzyme |
BamH1 +Pme1 | |
Subcloning
[edit]Date: 2011
Colony Screening
[edit]Date: 2012-1-6
Make miniprep and digest with BamH1 +Pme1
Result intepretation/Summary
[edit]- Sequencing Result:
Conclusion
[edit]