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3161 pET28b-GST-Myc-HA-Flag-V5 - Revision history
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Crm at 21:03, 20 March 2018
2018-03-20T21:03:02Z
<p></p>
<p><b>New page</b></p><div>Date: 3/4/2018<br />
<br />
Performed by:&nbsp;Paul<br />
<br />
subcloning<br />
<br />
[[File:3162 3164 3163 3161.JPG|RTENOTITLE]]<br />
<br />
miniprep digested with Nde1-Xho1<br />
<br />
[[File:3161 3165 947.JPG|RTENOTITLE]]<br />
<br />
Subcloning: Nde1+ Xho1<br />
<br />
Vector: 1803&nbsp;<span style="color: rgb(0, 0, 0); font-family: Arial, Verdana, sans-serif; font-size: 12px; background-color: rgb(251, 251, 239);">pCold-6His-GST-(E)-(3C)-PKARIIb</span> (insert is 1329bp)<br />
<br />
insert: 3159, 225 bp with Nde1 + Xho1<br />
<br />
3161:5-8<br />
<br />
Restriction enzymes:NdeI+XhoI<br />
<br />
'''Results: '''we sent 3021pminiT2 to do sequencing<br />
<br />
3161 is not good.<br />
<br />
[[File:Digestion of 3161 3021pMiniT2.JPG|RTENOTITLE]]<br />
<br />
'''Miniprep of 3168_3132_3161'''<br />
<br />
3168:1-4 BsrGI+NheI<br />
<br />
3132:5-8 BsrGI+NheI<br />
<br />
3161:9-12 NdeI+XhoI<br />
<br />
Buffer:Fastdigest<br />
<br />
Time:30Min<br />
<br />
Result:#7 seems good, so sent to do sequencing, the rest of them are suspicious<br />
<br />
[[File:Miniprep of 3168 3132 3161.JPG|RTENOTITLE]]<br />
<br />
Date:3/14/2018<br />
<br />
'''Miniprep digestion of 3131_3161_3167'''<br />
<br />
Name length Enzymen Buffer<br />
<br />
3131:1-4 342 BssHII+XhoI Cutsmart<br />
<br />
3161:5-8 225 NdeI+XhoI Cutsmart<br />
<br />
3167:9-12 397 BsrGI+NheI NEB 2<br />
<br />
Time:40min<br />
<br />
[[File:Miniprepof 3131 3167 3161.JPG|RTENOTITLE]]<br />
<br />
'''Miniprep digestion_3161_3168'''<br />
<br />
3161:1,2 NdeI+XhoI Buffer:Cutsmart<br />
<br />
3168:3 BsrGI+NheI<br />
<br />
Buffer:NEB 2<br />
<br />
Time:40min<br />
<br />
'''Result:seems good, do sequencing'''<br />
<br />
[[File:Miniprep of 3161 3168.JPG|RTENOTITLE]]<br />
<br />
'''Redo miniprep of 3131_3161_3167'''<br />
<br />
3131:1-4 BssHII+XhoI 342 Cutsmart<br />
<br />
3161:5-8 NdeI+XhoI 225 Cutsmart<br />
<br />
3167:9-12 BsrGI+NheI 397 NEB 2<br />
<br />
Time:40min<br />
<br />
[[File:Redo miniprep of 3131 3161 3167.JPG|RTENOTITLE]]<br />
<br />
== Restriction Digestion Analysis of 3159&nbsp;<span style="color: rgb(0, 0, 0); font-family: Arial, Verdana, sans-serif; font-size: 12px; white-space: nowrap;">pMiniT2-1FRG-HA-VL-kappa</span> ==<br />
<br />
by Paul, 3/16/2018<br />
<br />
Nde1 + Xho1 digestion:&nbsp;1803 (vetor)&nbsp; and 3159 (insert)<br />
<br />
[[File:1803 3159 Nde1 Xho1.JPG|RTENOTITLE]]<br />
<br />
Left: vector&nbsp;1803<br />
<br />
Right: 3159, there are too many bands, too complicated<br />
<br />
[[File:3159 EcoR1.JPG|RTENOTITLE]]<br />
<br />
Cut out EcoR1 band, middle ~ 1kb, purified with mini silica column, elute with 20 ul H2O<br />
<br />
digest&nbsp;with Nde1 + Xho1<br />
<br />
[[File:3159 1803 Nde1 Xho1.JPG|RTENOTITLE]]<br />
<br />
3/17/2018, cut out the smallest fragment, notice 1803 vector is overdigested, can not be used<br />
<br />
[[File:1803 3159 digestion.JPG|RTENOTITLE]]<br />
<br />
digest vector 1803 with Nde1-Xho1 (right lane)<br />
<br />
Date:3/19/2018<br />
<br />
Miniprep of 3161<br />
<br />
Restriction enzymes:NdeI+XhoI<br />
<br />
Buffer:Fastdigest<br />
<br />
TIme:30min<br />
<br />
Result:combine the rest of them except#2 to do sequence<br />
<br />
[[File:IMG 3623.JPG|RTENOTITLE]]<br />
<br />
Date:3/20/2018<br />
<br />
'''PCR screening of 3161'''<br />
<br />
Lane1,2:length 1300bp<br />
<br />
Primers:<br />
<br />
820-His-HA-His-T;<br />
<br />
T7-term(R)<br />
<br />
Lane 3: length 294<br />
<br />
Primers:pCold-seq-F;<br />
<br />
T7-term(R)<br />
<br />
[[File:IMG 3630.JPG|RTENOTITLE]]<br />
[[Category:Cloning]]</div>
Crm