Editing PlentiGP-App-303 (section)

= The following experiments were done by Haiyan<br>  =

-====================== 

== '''colonies screening for plentigp-app-303'''  ==

'''Purpose:''' colonies screening for plentigp-app-302 and pcr amplifying for the 295 (syn) and 306 (ef). 

'''Date: '''2011/08/01 

'''Notebook:''' 

'''Page:''' 

'''Clone ID:''' 

'''Experimenters:''' Haiyan, 

== '''Results'''  ==

=== Subcloning  ===

Date: 2011-8-01 

Digest pAAV-MCS (clone #254) with MLU1+ECORI <br> 

Digest 295 AND 306 PCR AMPLIFY (clone #) with MLU1+ECORI <br> [[Image:0802-ef1.JPG]]<br> 295 got two digest bands, something wrong, not continue. for the 306, too weak bands, not seen. just ligate the direct cut PCR product to see what is going on. <br> 

=== Colony Screening for app  ===

Date: 2011-8-1<br> 

Make miniprep and digest with Ecori+agei<br> 

[[Image:0801-app.JPG]] <br> 

Lane 1: Marker <br> Lane 2: 295 PCR 10UL <br> Lane 3:306 PCR 10UL <br> Lane 4-9, pLVGP-app, Ecori+agei<br> lane 5 and 8 should be positive clone. <br> 

<br> 

Date: 2011-8-3<br> 

[[Image:0803-app.JPG]] <br> 

Lane 1: Marker <br> Lane 2-8: pLentigp-app, Ecori+agei<br> lane 3, 6 and 8 should be positive clone. <br> lane 9, plenti-pka-gluc, nhei+xhoi; should be positive clone.<br> 

<br> 

Date: 2011-8-5<br> 

[[Image:0806-app1.JPG]] <br> 

Lane 1: Marker <br> Lane 2, app amplify,305 (original app DNA )app-f/app-r primer set, pfusion enzyme, 10ul run gel (50ul volume);<br>Lane 3&amp;4, pka 1 and 2 amplify, nhei-f1/xhoi1 and nhei-f2/xhoi2 primer set, pfusion enzyme, 10ul run gel (50ul volume);<br> Lane 5-10, colonies amplify using app-f/app-r primer set<br> Lane 11, 305 (original app DNA )app-f/app-r primer set <br>; 

[[Image:0806-app2.JPG]] <br> Lane 1-3 overlap with last fig 9-11 <br>; lane 3-8, app plasmid as template; names 305, app 1-5; <br>, regular taq could not get strong pcr amplify bands for app,app-f/app-r primer set <br> lane 4, app1 DNA<br> lane 5, app2<br>lane 6, app3<br>lane 7, app4<br>lane 8, app5<br>lane 9, plentiGP ECORI+AgeI cut <br> lane 10, app 1 ECORI+AgeI cut, same plasmid with lane 4 <br> 

[[Image:0806-app3.JPG]] <br> lane 1, marker <br> lane 2, app 2 ECORI+AgeI cut, same plasmid with last fig lane 5;<br>lane 3, app 3 ECORI+AgeI cut, same plasmid with last fig lane 6;<br>lane 4, app 4 ECORI+AgeI cut, same plasmid with last fig lane 7;<br>lane 5, app 5 ECORI+AgeI cut, same plasmid with last fig lane 8;<br>lane 6,plentiGP bamhi+nhei cut <br>lane 7, app 1 bamhi+nhei cut<br>lane 8, app 2 bamhi+nhei cut<br>lane 9, app 3 bamhi+nhei cut<br>lane 10, app 4 bamhi+nhei cut<br>lane 11, app 5 bamhi+nhei cut<br> 

conclusions: seems plentigp DNA already degraded in 4 degree; <br> it seems app 2 and app3 are positive clones, colonies screening from app 2 transform plates; app 2 is yesterday's sequence clone, 483 ng/ul, ratio is 1.83 <br> 

<br> 

<br> 

== '''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:<br>

== '''Conclusion'''  ==

Restriction digestion <br> 

shows&nbsp; pLentiGP clone #1 may be good, <br> 

pLVGP-PKAalpha 3+4 is good<br> 

===  ===

[[Category:Cloning]]