Hylei at 17:32, 22 August 2011

2011-08-22T17:32:52Z

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== '''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, Ruili

== '''Results''' ==

=== Subcloning ===

=== Test whether the 299_plentigp_gluc is the good vector for the 301_gluc_pka === Date: 2011-08-08

 [[Image:0808 app pcr 0808.JPG]] 

Lane 7 &8 : 299_plentigp_gluc as tmp, cmv-f/gluc-r primer set, pcr amplify 

explaination: two plasmids both have the amplified bands, the vector should no problem. 

Date: 2011-08-08

[[Image:0808 gluc vector 0808.JPG]] 

Lane 1-4: 299_plentigp_gluc vector 1, 2, 3 and 219_plentigp nhei+xhoi 

Lane 5-8: 299_plentigp_gluc vector 1, 2, 3 and 219_plentigp nhei+bamhi 

299_plentigp_gluc vector is still the good vector, lane 1 ligate with the annealed pka to do transform

 

=== Colony screening === 

 Date: 2011-08-10

 [[Image:IMG 1253.JPG]] CMV-F/pka-xhoi-r primer for colonies screening, 12 colonies, other 2 colonies in other figs, same band with colonies 3, 4. seems lane 7 has the longer band, others has the short band. Grow lane 6,7,8,9 to do miniprep.

 

Date: 2011-08-11

 [[Image:0811-pka.JPG]] Lane 1: Marker Lane 2, pka 6 (above lane 6) bamhi+xhoi Lane 3, pka 7 (above lane 7) bamhi+xhoi Lane 4, pka 8 (above lane 8) bamhi+xhoi Lane 5, pka 9 (above lane 9) bamhi+xhoi Lane 6, pka-gluc (vector for pka-gluc) bamhi+xhoi Lane 7, pka-gluc (vector for pka-gluc) bamhi+xhoi Lane 8, pka 6 (above lane 6) nhei+xhoi Lane 9, pka 7 (above lane 7) nhei+xhoi Lane 10, pka 8 (above lane 8) nhei+xhoi Lane 11, pka 9 (above lane 9) nhei+xhoi Lane 12, pka-gluc (vector for pka-gluc) nhei+xhoi Lane 13, pka-gluc (vector for pka-gluc) nhei+xhoi 

we think lane 3 (clone 7) should be positive clone. 

after sequencing, clone 7 has lot mutation, could not use 

2011-08-19 

[[Image:0819.jpg]] lane 1-2: pet 28b-lss-mkate 2 (229) bamhi+xhoi lane 3: plentigp-gluc (299) nhei+xhoi lane 4: pET3a-Muts (352) ndei+ecoi lane 5: pET3a-Muts (352) ndei+hindiii lane 6: pET28b ndei+ecoi lane 7: pET28b ndei+hindiii lane 8: pdbhisMBP ndei+ecoi lane 9: pdbhisMBP ndei+HINDIII Lane 10: pET3a-Muts (352) tmp, pcr amplify lane 11-13, pka1, primer :PKAalpha-Nhe1-F1/PKAalpha-6His-Xho1-R1, tmp pkaalpha lane 14-16: pka2, primer :PKAalpha-Nhe1-F2/PKAalpha-6His-Xho1-R2, tmp pkaalpha for the pcr amplify, fusion enzyme, 95, 68 3min extension. 

Date: 2011-7-13

[[Image:Pka+gluc.JPG]] 

Lane 1: pka1, primer :PKAalpha-Nhe1-F1/PKAalpha-6His-Xho1-R1, tmp pkaalpha 

Lane 2: pka2, primer :PKAalpha-Nhe1-F2/PKAalpha-6His-Xho1-R2, tmp pkaalpha 

Lane 3: gluc,primer :GLuc1 BamH1 A8/Gluc-Nhe1-R Nhe1, tmp: pET28b-Gluc (ID: 301) 

Phusion enzyme, 25 cycle

digest plentiGP with Nhei/Xhoi (for pka) and plentiGP with Nhei/Bamhi (for Gluc)

anneal the pka pcr fragment and ligate with plentiGP with Nhei/Xhoi; Nhei/Bamhi digest the Gluc PCR fragment and ligate with plentiGP with Nhei/Bamhi transform STBL3 

 

=== Colony Screening ===

Date: 2011-7-15 

 [[Image:App.JPG]] Lane 1: Marker Lane 2+3: gluc,primer :GLuc1 BamH1 A8/Gluc-Nhe1-R Nhe1, tmp: pET28b-Gluc (ID: 301), NheI+BamHI Lane 4: plentiGP with Nhei/Bamhi (for Gluc) Lane 5+6: App,primer :AmyloidBP-F/AmyloidBP-r, tmp: pCMV-SPORT6-App (ID: 305), NheI+BamHI Lane 7: plentiGP with Nhei/Bamhi (for app) Lane 8: plentiGP with Nhei/Xhoi (for pka) Phusion enzyme, 25 cycle ligate 2+3 with 4' 5+6 with 7. anneal the pka pcr fragment and ligate with plentiGP with Nhei/Xhoi; Nhei/Bamhi digest the Gluc PCR fragment and ligate with plentiGP with Nhei/Bamhi transform STBL3 2011-7-16 [[Image:App+gluc.JPG]] Lane 1: Marker Lane 2: plentiGP with Nhei/Bamhi Lane 3 &4: app pcr amplify, cut with Nhei/Bamhi Lane 5 to Lane 8: plenti-gluc cut with Nhei/Bamhi seq Lane 5 ligate Lane 2 with Lane 3&4. 

2011-7-16 PCR Amplify to screen colonies for pet30-his, primer set: T7R/Pet 8 His-s [[Image:Pet 30 -his.JPG]] Lane 1: Marker Lane 2: colonie 1 Lane 3: colonie 2 Lane 4: colonie 3 Lane 5: colonie 4 Lane 6: pet 30b template to amplify lane 2,3,5 should be positive; seq lane 2; 

 2011-7-25 colonies screen for 303_pLentiGP-App [[Image:App+pka.JPG]] Lane 1: pka1, primer :PKAalpha-Nhe1-F1/PKAalpha-6His-Xho1-R1, tmp pkaalpha 

Lane 2: pka2, primer :PKAalpha-Nhe1-F2/PKAalpha-6His-Xho1-R2, tmp pkaalpha Lane 3: 299_plentiGluc NCOI+XHOI digestion Lane 4-8: colonie screening for 303_pLentiGP-App 

seq lane 4 failed, try seq lane 8 again; 

 2011-7-25 subclone 256, 241 cre to pet8his [[Image:Cre+p.JPG]] Lane 1: Marker 

Lane 2,3: 256_pAAV-cre Ncoi+xhoi Lane 4,5: 241 plentigp-cre NCOI+XHOI digestion Lane 6,7: 286 pet 8His NCOI+XHOI digestion 

ligate 2,3 with lane 6; ligate 4,5 with lane 7; 

2011-07-25 colonies screening for 300_plenti-gluc-pka and pet8his-cre [[Image:Pka+cre-0725.JPG]] Lane 1: Marker Lane 2-8: plenti-gluc-pka colonies 1-7 nhei+xhoi Lane 9-13: 256_pAAV-cre Ncoi+xhoi cut and ligate with pet8his cut Lane 14-18: 241_pAAV-cre Ncoi+xhoi cut and ligate with pet8his cut seq lane 13 

 

 2011-07-26 colonies screening for 300_plenti-gluc-pka [[Image:Pka-0725.JPG]] Lane 1: Marker Lane 2-8: plenti-gluc-pka colonies 1-7 nhei+xhoi Lane 9-12: plenti-gluc-pka colonies 1-7 pcr amplify, using gluc1-a8 and pka-xhoi r primer for other 3 colonies, got same pcr amplify band, data not showing It is possible Nhei site does not work now, but has the pka insert. 

2011-07-27 miniprep kit comparison [[Image:Kit comparison.JPG]] 5ul/50ul run gel, Kit order: Qiagen, Original, Omega, introbio Lane 1: Marker Lane 2-5: dsAAV_SMN1, lane 2, 30ng/ul, 260/280 is 2.02; lane 3, 330ng/ul, 260/280 is 1.19; lane 4, 10ng/ul, 260/280 is ---; lane 5, 40ng/ul, 260/280 is 1.3; Lane 6-9: dsAAV_SMN2, 

Lane 10-13: plenti-app, 

 

2011-07-27 medium prep DNA quality check and pka-gluc colonies check [[Image:Medium prep-0726.JPG]] Lane 1: Marker lane 2, pAAV-MCS lane 3, Pet8His; lane 4, pAAV-CRE lane 5, PLENTI-MKATE2 lane 6, PKA-GLUC BAMHI+XHOI lane 7, PKA-GLUC NHEI+XHOI lane 8, PKA-GLUC BAMHI+NHEI Throw away lane 4,5; seems no positive for pka-gluc. 

2011-07-29 PCR check app colonies and pka-gluc colonies check [[Image:0729-app.JPG]] 

Lane 1: Marker lane 2-7, cmv-f/amyloid-r pcr amplify for colonies 1-6 lane 8-9, bghr/amyloid-f pcr amplify for colonies 1-2, plus the following picture; [[Image:0729-pka.JPG]] 

 lane 1-6, bghr/amyloid-f pcr amplify for colonies 1-6, overlap with the upper picture lane 7-9, PLENTI-pka-gluc nhei+xhoi lane 9 positive clone, seq 

 

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

== '''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]]

Plenti-gluc-pka-301 - Revision history

Hylei at 17:32, 22 August 2011