PlentiGP-App-303

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

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

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

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

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

The following experiments were done by Haiyan

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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

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

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

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

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
;

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

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