Editing Plenti-gluc-pka-301 (section)

=== 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> 

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