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163 pT7A3-MBP-(Tev)-GST-(3C)-sfGFP - Revision history
2026-10-18T12:56:45Z
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https://eln.excellgen.com/index.php?title=163_pT7A3-MBP-(Tev)-GST-(3C)-sfGFP&diff=19782&oldid=prev
Crm at 15:45, 2 January 2019
2019-01-02T15:45:24Z
<p></p>
<p><b>New page</b></p><div><pre>PCR fragment 1 from 2369 pT7A3-lc-MBP-(TEV)<br />
2094-T<br />
CTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATAT<br />
TTTATTAAAACAAATTGAAATTCTTCCTCTATATAGTAC<br />
<br />
MBP-seq-R (CGGTTCTTGCAGGTTGAAC)<br />
gttcaacctgcaagaaccg<br />
<br />
PCR fragment 2 from 2369 pT7A3-lc-MBP-(TEV)<br />
<br />
pET-seq-F2 ATCTCGACGCTCTCCCTTATG<br />
2094-B<br />
CATGATATATCTCCTTCTTAAAGTTAAACAAAATTATTT<br />
<br />
Purify on gel, PCR again <br />
pET-seq-F2 ATCTCGACGCTCTCCCTTATG<br />
MBP-seq-R (CGGTTCTTGCAGGTTGAAC)<br />
gttcaacctgcaagaaccg<br />
<br />
Digest with BsaI + SphI<br />
clone into BsaI + SphI 161 pT7A3-MBP-(Tev)-GST-(3C)-sfGFP</pre><br />
<br />
[[File:163 PCR.JPG|RTENOTITLE]]<br />
<br />
[[File:163 165 v digestion.JPG|RTENOTITLE]][[File:164 digestion.JPG|RTENOTITLE]]<br />
<br />
[[File:163 digestion.JPG|RTENOTITLE]]<br />
<br />
#1, #5 looks good<br />
<br />
Sequencing result: it does not have T7A3 promoter, It is cold promoter.. it is clone 160<br />
<br />
[[File:164 pJet digestion.JPG|RTENOTITLE]][[File:162 160 164 pJet digestion.JPG|RTENOTITLE]]<br />
<br />
[[File:163 164 PCR Screening.JPG|RTENOTITLE]]http://www.excellgen.net/eln/images/2/2c/163_164_digestion.JPG<br />
<br />
<br />
<br />
[[File:164 163 XbaI Xho1.JPG|RTENOTITLE]][[File:164 163 Nhe1 Xho1.JPG|RTENOTITLE]]<br />
<br />
[[File:164 163 Xba Xho1 2.JPG|RTENOTITLE]]<br />
<br />
== Redo ==<br />
<br />
1/1/19: The above methods were not working<br />
<br />
use subcloning method:<br />
<br />
digest 167 pT7A3-MBP-TRX-6H #2 with Sph1+ BsaI<br/>digest 161 pT7A3-MBP-(Tev)-GST-(3C)-sfGFP&nbsp; with Sph1+ BsaI<br />
<br />
[[File:167 161 316 160 digestion.JPG|RTENOTITLE]][[File:316 167 digestion.JPG|RTENOTITLE]]<br />
<br />
<br />
<br />
163-1 from 54ng/ul digestion<br />
<br />
163-2 from 34ng/ul digestion<br />
<br />
[[File:163 digestion 1 1 19.JPG|RTENOTITLE]][[File:162 163 digestion2.JPG|RTENOTITLE]]<br />
<br />
163-2 #1, pellet not green, all other clones are green<br />
[[Category:Cloning]]</div>
Crm