Hylei at 20:18, 8 August 2011

2011-08-08T20:18:44Z

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== '''Cloning of App to pLentiGP''' ==

'''Purpose:''' Cloning of App to pLentiGP

'''Date: '''2011/08/5 to 2011/8/7

'''Notebook:'''

'''Page:'''

'''Clone ID: '''303

'''Experimenters:''' Paul

== '''Results''' ==

=== PCR ===

Date: 2011-8-05

Condition: 

PCR was run by Haiyan 

[[Image:App PCR amplification 2 2011 8 5.jpg]] 

cut out band as shown above, gel purified, elute with 20 ul water. 

=== Digestion and Gel Purification ===

Date: 2011-8-5 

Digested purified DNA and vector plasmid pLentiGP (219) with 0.5 ul BamH1 + 0.5 ul Nhe1 

run 1% gel 

[[Image:App gel purification after digestion 2011 8 7.jpg]] 

cut out lane 1 and 2 bands (APP) and vector (lane 3). 

lane 4 is 1 kb ladder 

mix gel slice, add 0.8 ml QG buffer, purified using qiagen spin column, elute with 20 ul water. 

add 2.5 ul ligation buffer, 0.5 ul ligase, ligate at 25 oc for 2 hours. 

transform STBL3 

plate on LB/amp plate o/n at 37oC. 

 

=== Colony Screening ===

Date: 2011-8-6 

pick up 7 colonies, grow in 5 ml LB/amp O/N at 37 oC 

Date: 2011-8-7 

Make miniprep elute with 50 ul EB, digest 3 ul DNA with 0.25 ul BamH1 + Nhe1, 1.5 ul 10X fastdigest buffer in 15 ul final vol, 37 oC for 45 min. 

Run 1% gel 

 [[Image:App colony screening restriction digestion 2 2011 8 7.jpg]] 

Lane 1 & 12, 1 kb ladder 

Lane 2 to 8, pLentiGP-app digested with BamH1 + Nhe1 

Lane 9 pLentiPlus digested with Nhe1+ Nde1 

Lane 10 pLentiPlus digested with BamH1+ Nde1 

Lane 11 pLentiPlus digested with Xho1+ Nde1 

Looks like lane 2, 5, 6, 7, 8 (clone 1,  4, 5, 6, 7) are good, discard colonies 2 and 3 (lane 3 and 4). 

vector pLentiPlus is also good.

=== Additional PCR Screening to test the above app colonies ===

Date: 2011-8-8 

using cmv-f/app-r primer set to check the above clone 1,  4, 5, 6, 7 

Run 1% gel 

 [[Image:0808_app_pcr_0808.JPG]] 

Lane 1 ; 1 kb ladder 

Lane 2 to 6, clone 1,  4, 5, 6, 7 pcr amplify 

Looks like lane 2-6 have amplified bands (clone 1,  4, 5, 6, 7) 
seq clony #5 (lane 3).

Haiyan, 2011-08-08 

== '''Result intepretation/Summary''' ==

'''Digestion''': Clone 1, 4, 5, 6, 7 may be  good. 

'''Sequencing''':send plasmid from clony #5 for sequencing 

 

== '''Conclusion''' ==

 

= The following experiments were done by Haiyan =

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

== '''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 

Digest 295 AND 306 PCR AMPLIFY (clone #) with MLU1+ECORI [[Image:0802-ef1.JPG]] 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. 

=== Colony Screening for app ===

Date: 2011-8-1 

Make miniprep and digest with Ecori+agei 

[[Image:0801-app.JPG]] 

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

 

Date: 2011-8-3 

[[Image:0803-app.JPG]] 

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

 

Date: 2011-8-5 

[[Image:0806-app1.JPG]] 

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

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

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

conclusions: seems plentigp DNA already degraded in 4 degree; 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 

 

 

== '''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 

=== ===

[[Category:Cloning]]

PlentiGP-App-303 - Revision history

Hylei at 20:18, 8 August 2011