Editing Plenti-gluc-pka-301 (section)

== '''Results'''  ==

=== Subcloning  ===

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

<br> [[Image:0808 app pcr 0808.JPG]] <br> 

Lane 7 &amp;8&nbsp;: 299_plentigp_gluc as tmp, cmv-f/gluc-r primer set, pcr amplify<br> 

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

Date: 2011-08-08 

[[Image:0808 gluc vector 0808.JPG]] <br> 

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

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

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

<br> <br> 

=== Colony screening ===<br> 

<br> Date: 2011-08-10 

<br> [[Image:IMG 1253.JPG]] <br> 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. 

<br> <br> 

Date: 2011-08-11 

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

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

after sequencing, clone 7 has lot mutation, could not use <br> 

2011-08-19 <br> 

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

Date: 2011-7-13 

[[Image:Pka+gluc.JPG]] <br> 

Lane 1: pka1, primer&nbsp;:PKAalpha-Nhe1-F1/PKAalpha-6His-Xho1-R1, tmp pkaalpha<br> 

Lane 2: pka2, primer&nbsp;:PKAalpha-Nhe1-F2/PKAalpha-6His-Xho1-R2, tmp pkaalpha<br> 

Lane 3: gluc,primer&nbsp;:GLuc1 BamH1 A8/Gluc-Nhe1-R Nhe1, tmp: pET28b-Gluc (ID: 301) <br> 

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; <br> Nhei/Bamhi digest the Gluc PCR fragment and ligate with plentiGP with Nhei/Bamhi<br> transform STBL3 <br> <br> 

<br> 

=== Colony Screening  ===

Date: 2011-7-15<br> 

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

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

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

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

seq lane 4 failed, try seq lane 8 again;<br> 

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

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

ligate 2,3 with lane 6; ligate 4,5 with lane 7;<br> 

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

<br> 

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

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

Lane 10-13: plenti-app,<br> 

<br> 

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

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

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

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

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