Multiple clones pAd5

SubCloning of fluorescent fragments to pAd5-MCS (280)

Purpose: SubCloning of fluorescent fragments to pAd5-MCS (280).

Date: 2011-8-23

Notebook:

Page:

Clone ID:

Experimenters: Haiyan

Results

Subcloning

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

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

• 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

Restriction digestion

shows  pLentiGP clone #1 may be good,

pLVGP-PKAalpha 3+4 is good

Paul.xu 08:23, 15 June 2011 (EST)