300 PLentiGP-Gluc-PKA-Alpha

SubCloning of PKA alpha to pLentiGP

Purpose: Subcloning of PKA alpha to pLentiGP for expression of PKA in 293 cells with GFP screening.

Date: 2011-6-8 to 2011-6-16

Notebook:

Page:

Clone ID:

Experimenters: Paul, Haiyan,

Results

Sequencing

pLentiGP-Gluc was sequenced

PCR

Haiyan did PCR to amplify PKAalpha

PCR:
1. PKAalpha-Nhe1-F1 + PKAalpha-6His-Xho1-R1
CTAGC atgggcaacg ccgccgccgc c-----PKA 6XHis-TAA C
GATCG tacccgttgc ggcggcggcg g ATT G

2. PKAalpha-Nhe1-F2 + PKAalpha-6His-Xho1-R2
C atgggcaacg ccgccgccgc c---PKA -- 6XHis-TAA ctcga
G tacccgttgc ggcggcggcg g ATT gagct

Annealing:

Mix PCR 1 and 2 in Qiagen PB buffer (or Qiagen PCR purification buffer),
load to Qiagen miniprep spin column, wash with 2X of 350 ul PE,
elute with 25 ul H20, add 2.5 ul 10X PCR buffer,
heat at 95 oC for 2 min, turn off PCR machine, cool slowly to 25 oC.

Gel purify, then ligate to Nhe1+Xho1 cut pLentiGP-Gluc

Pick colones: 5, 7, 9, 11, 13

grow in LB/AMP

Make Miniprep

Digestion with Nhe1+ Xho1

DNA conc using nanodrop

Clone	conc	260/280
5	114	1.89
7	127	1.91
9	136	1.91
11	120	1.91
13	132	1.92

--Paul.xu 15:06, 27 August 2011 (EST) Sequencing: #13, make 10 ng/ul, 10 ul, using pGreenPuro-Seq-R as primer

Subcloning

Date: 2011-6-8

Digest pLVGP (clone #) with NdeI + BamH1

Digest pLVX-Plus2-PKAalpha (clone #) with NdeI + BamH1

cut out insert and vector, ligate and transform STBL3

Colony Screening

Date: 2011-6-15

Make miniprep and digest with NdeI + BamH1

Lane 1: pLVGP-PKAalpha 3+4, Xho1 + Mlu1

Lane 2: pLentiGP #1, Xho1 + Mlu1

Lane 3: pLentiGP #6, Xho1 + Mlu1

Lane 4: pLVGP #1, Xho1 + Mlu1

Lane 5: pLVGP-PKAalpha 3+4, Nde1+ BamH1

Lane 6: pLentiGP 1, Nde1+ BamH1

Lane 7: pLentiGP 6, Nde1+ BamH1

Lane 9: pLVGP1, Nde1+BamH1

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)