Paul.xu: moved 300 PLentiGP-PKA-Alpha to 300 PLentiGP-Gluc-PKA-Alpha

2011-09-01T02:09:05Z

moved 300 PLentiGP-PKA-Alpha to 300 PLentiGP-Gluc-PKA-Alpha

New page

== '''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 

 

=== PCR screening ===

=== [[Image:300_pLentiGP-Gluc-PKAalpha_PCR_screening_8_26_2011.jpg]] ===

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

grow in LB/AMP 

Make Miniprep 

Digestion with Nhe1+ Xho1 

[[Image:300_pLentiGP_Gluc_PKAalpha_restriction_digestion_8_27_2011.jpg]] 

DNA conc using nanodrop 

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| Clone 
| conc 
| 260/280 
|-
| 5 
| 114 
| 1.89 
|-
| 7 
| 127 
| 1.91 
|-
| 9 
| 136 
| 1.91 
|-
| 11 
| 120 
| 1.91 
|-
| 13 
| 132 
| 1.92 
|}

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

 

 

=== the following are old exp ===

=== 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 

[[Image:PLentiGP-PKAalpha 6 14 2011 2.JPG]]

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 

=== ===

[[User:Paul.xu|Paul.xu]] 08:23, 15 June 2011 (EST) 

[[Category:Cloning]]

300 PLentiGP-Gluc-PKA-Alpha - Revision history

Paul.xu: moved 300 PLentiGP-PKA-Alpha to 300 PLentiGP-Gluc-PKA-Alpha