Multiple clones pAd5
SubCloning of fluorescent fragments to pAd5-MCS (280)
[edit]Purpose: SubCloning of fluorescent fragments to pAd5-MCS (280).
Date: 2011-8-23
Notebook:
Page:
Clone ID:
Experimenters: Haiyan
Results
[edit]Subcloning
[edit]Date: 2011-8-23
Release fragments with Xhoi + BamH1
Digest pAd5-MCS (clone #280) with Xhoi + BamH1
cut out insert (1-9) and vector (10, 11), ligate and transform STBL3
| lane | 1 | 2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
13 |
||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| plasmid | lss-mkate2 | IFP |
RFP |
eEGFP |
NEPTUNE |
BFP |
YFP |
mkate2 |
CFP |
280 |
280 |
plenti-YFP1 |
plenti-YFP2 |
lane 12, 13 colonies screenign fro plenti-YFP
lane 14, 15 colonies screenign fro pAAV-YFP
Colony Screening
[edit]Date: 2011-8-26
Make miniprep and digest with xhoi + BamH1
Lane 1-4: pAd5-LSS-mkate2 (365) bamhi+xhoi
Lane 5-8: pAd5-IFP (234) bamhi+xhoi
Lane 9-12: pAd5-RFP (230)bamhi+xhoi
Lane 13-16: pAd5-EGFP (304)bamhi+xhoi
Pick up 1, 6, 9, 15 to freeze DNA and bacteria.
Lane 17-20: pAd5- Neptune (366) bamhi+xhoi
Lane 21-24: pAd5-BFP (233) bamhi+xhoi
Lane 25-28: pAd5-YFP (231)bamhi+xhoi
Lane 29-31: pAAV-mKate2 (228)bamhi+xhoi
Lane 32: pAd5-cfp (232)bamhi+xhoi
Pick up 17, 22, 26, 30, 32 to freeze DNA and bacteria.
Result intepretation/Summary
[edit]- PKAalpha 3+4 has one internal Xho1 site. GFP-Puro can be cut out with Xho1 + Mlu1. So Lane 1 has correct number of bands.
- PKA alpha 3+4 with CMV promoter can be cut out with Nde1+ BamH1. Lane 5 has correct number of bands.
- lane 6 maybe good (incomplete digestion)
- Sequencing Result:
Conclusion
[edit]Restriction digestion
shows pLentiGP clone #1 may be good,
pLVGP-PKAalpha 3+4 is good
Paul.xu 08:23, 15 June 2011 (EST)