2015-pEC2
Cloning of 2015-pEC2
[edit]Purpose: Cloning of 2015-pEC2
Date: 2014-2-23---2014-2-28
Notebook:0
Page:
Clone ID:2015
Experimenters: yingying
Results
[edit]cloning
[edit]
pcr1
Fragment 1 pUC19 as template
M13-F-pUC19-F+pUC19-HindIII-R
pcr2
Fragment 2 pUC19 as template
pUC19-HindIII-F+pUC19-BglII-R
pcr3
gene synthesis
1 ul of 10 uM BglII-T7-Term
1 ul of 1 uM of the following oligos
pEC2-1
pEC2-2
pEC2-3
pEC2-4
1 ul of 10 uM pFastBacF-Nco1-6H-Nde1-R
pcr4
pEC as template
pFastBacF-Nco1-6H-Nde1-F+M13-F-pUC19-R
PCR:Gel purify these 4 fragments
mix, and PCR again using
BglII-T7-Term+pUC19-BglII-R
| lane |
3 |
| PCR | |
| BglII |
the final PCR product should be 2.5 kb.
digest with BglII, purify and ligate.
Colony Screening
[edit]Date: 2014-2-28
Make miniprep and digest with
Digest with HindIII alone, run on EB gel: should get 1kb and 1.5 kb fragments
Result intepretation/Summary
[edit]Conclusion
[edit]