Plenti-gluc-pka-301

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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;

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

Conclusion

Restriction digestion

shows  pLentiGP clone #1 may be good,

pLVGP-PKAalpha 3+4 is good

Paul.xu 08:23, 15 June 2011 (EST)