2222 pFastbacDual-VSVG-M-6H-Tat-Cre-MFGE8-Gag-Cre
Cloning of 2222 pFastbacDual-VSVG-M-6H-Tat-Cre-MFGE8-Gag-Cre
[edit]Purpose: Cloning of 2222 pFastbacDual-VSVG-M-6H-Tat-Cre-MFGE8-Gag-Cre==
Date: 2014-6----
Notebook:0
Page:
Clone ID:2222
Experimenters: shanyi
Results
[edit]CLONING
[edit]1. PCR p10 promoter from pFastbac-Daul
p10-Not1-F
gctac GCGGCCGC GAGTATACGGACCTTTAATTC
p10-Sal1-Gag-R
(ATTTACAATCACTCGACGAAGACTGTCGACatgggtgcgagagcgtcagtat)
atactgacgctctcgcacccatGTCGACAGTCTTCGTCGAGTGATTGTAAAT
2. PCR HIV Gag-Cre from 1968 pCMV5-Gag-p2b-NLS-Cre-WPRE-oriP
p10-Sal1-Gag-F
ATTTACAATCACTCGACGAAGACTGTCGACatgggtgcgagagcgtcagtat
iCre-Xba1-R
(CTGCTGGAAGATGGCGATTAG TCTAGA tagcta)
tagctaTCTAGACTAATCGCCATCTTCCAGCAG
3. PCR again to join two fragments (maybe it is not necessary to purify two fragments on gel)
p10-Not1-F
gctac GCGGCCGC GAGTATACGGACCTTTAATTC
iCre-Xba1-R
(CTGCTGGAAGATGGCGATTAG TCTAGA tagcta)
tagctaTCTAGACTAATCGCCATCTTCCAGCAG
4. Digest with Not1 + Xba1 and clone into Not1 + Xba1 digested 2221 pFastbacDual-p10-VSVG-M-6H-Tat-Cre-MFGE8
Colony Screening
[edit]Date: 2014
Make miniprep,
