280 pAd5-CMV-MCS
(Redirected from PAd5-CMV-MCS)
Convert pAd5-CMV-IRES-GFP (227) to pAd5-CMV-MCS (280)
[edit]Purpose: convert pAd5-CMV-IRES-GFP to pAd5-CMV-MCS
Date: 8-18-2011
Notebook:Paul
Page: x
Clone ID: 280
Experimenters: Paul
Results
[edit]Digestion
[edit]Date: 2011-8-18
Remove GFP
Digest pAd5-CMV-IRES-GFP (227) with KpnI + NotI, cut out vector (not two small fragments), gel purify,
Ligate with ligate with annealed pAD5-MCS-T3 + pAD5-MCS-B3, for 1 hour at RT, transform STBL3 with 10 ul ligation mix.
Colony Screening
[edit]Date: 2011-8-20
Make miniprep and digest with Xba1+ Nde1
cut our promoter (partly) using NdeI + XbaI, Xba1 is introduced by oligos (pAD5-MCS-T3 + pAD5-MCS-B3)
Result intepretation/Summary
[edit]- clone # 1, 2, 4 look good
- Miniprep OD260
| ng/ul | 260/280 | |
| #1 | 7.5 | 1.72 |
| #2 | 11 | 2.1 |
| #4 | 15 | 1.9 |
- sequencing, #4
Conclusion
[edit]
--Hylei 15:35, 20 August 2011 (EST)