https://eln.excellgen.com/index.php?action=history&feed=atom&title=2421-pEC-Thaumatin2421-pEC-Thaumatin - Revision history2026-10-18T05:16:17ZRevision history for this page on the wikiMediaWiki 1.44.0https://eln.excellgen.com/index.php?title=2421-pEC-Thaumatin&diff=14479&oldid=prevExcellgen at 20:52, 28 May 20152015-05-28T20:52:02Z<p></p>
<p><b>New page</b></p><div>== '''Cloning of <span dir="auto">2421-pEC-Thaumatin</span>''' ==<br />
<div>'''Date:&nbsp;''' 2015-4<br/></div><br />
'''By: Xin'''<br />
<br />
'''Results:'''<br />
<br />
Gene Synthesis: about 750bp. The vector PEC (1454)<br />
<br />
'''[[File:2421, 2418, 2434 clone 4-16-15.JPG|313x151px|RTENOTITLE]]'''<br />
<br />
== ==<br />
<br />
&nbsp; Digested by NdeI /KpnI, ligation, transformation.<br />
<br />
== '''Screening:''' ==<br />
<br />
ini-preparation<br />
<br />
[[File:2421screening.JPG|RTENOTITLE]]<br />
<br />
== ==<br />
<br />
Send #2 to sequence on April 23,2015<br />
<br />
Send #3 to sequence on April 25, 2015<br />
<br />
'''''Fix''''' #3 on April 26, 2015:<br />
<br />
use #3 sequenced on (April 25, 2015) as template,<br />
<br />
PCR fragment I: the oligos 2421-Thaumatin-1 and 2421-Thaumatin-4<br />
<br />
== ==<br />
<br />
PCR fragment II: the oligos 2421-Thaumatin-5 and 2421-Thaumatin-18<br />
<br />
[[File:2423,2436,2421 PCR.JPG|343x143px|RTENOTITLE]]<br />
<br />
Join two fragments together.<br />
<br />
[[File:2436, 2423, 2421 PCR.JPG|416x155px|RTENOTITLE]]<br />
<br />
== ==<br />
<br />
'''Screening''':<br />
<br />
[[File:1916 2423 2421 screening.JPG|475x152px|RTENOTITLE]]<br />
<br />
&nbsp;1,2,3 and 4 colonies are positive.<br />
<br />
== ==<br />
<br />
Send #4 to sequence on 5/5/2015, the sequencing is still same as the template.<br />
<br />
== ''Fix 5/5/2015'' ==<br />
<br />
The template used#2&nbsp; (sequenced on April 23,2015)<br />
<br />
The mistakes was happened on oligo 2421-Thaumatin-1 / -2&nbsp;<br />
<br />
There are two ways to fix:<br />
<br />
One is to use 2421-Thaumatin-1 and -18 redo a PCR.<br />
<br />
== ==<br />
<br />
[[File:2412.JPG|391x135px|RTENOTITLE]]<br />
<br />
The second way to fix is to do a PCR use 2421-Thaumatin-3 and -18.<br />
<br />
[[File:2421;1916.JPG|213x189px|RTENOTITLE]]<br />
<br />
== ==<br />
<br />
Then use the PCR template to add 1umol 2421-Thaumatin-2 and use the primer is 2421-Thaumatin-1 and -18 do a big PCR.<br />
<br />
[[File:1916,2421.JPG|193x144px|RTENOTITLE]]<br />
<br />
== ==<br />
<br />
=== '''Screening''': ===<br />
<br />
screening 4 colonies first (from Fix method one plate)<br />
<br />
[[File:2421 5-10-15.JPG|331x201px|RTENOTITLE]]<br />
<br />
== ==<br />
<br />
Send #4 to sequence, the result is still same as previous.<br />
<br />
&nbsp;Rescreening 8 colonies, 1-4 are the colonies conducted by fix method one; 5-8 are the colonies conducted by fix method two.<br />
<br />
[[File:2421,2412.JPG|402x181px|RTENOTITLE]]<br />
<br />
== ==<br />
<br />
Send sequencing #6 to sequencing. There is still one mistake, which is different as previous mistakes.&nbsp;<br />
<br />
Rescreen 7 colonies (from fix method II plate).<br />
<br />
&nbsp;[[File:2421 screening 5-15-15.JPG|296x157px|RTENOTITLE]]<br />
<br />
Send #2 for sequencing on 5/15/2015.<br />
<br />
== ''Fix on 5/28/2015'' ==<br />
<br />
Use the sequencing on 5-15-2015 as template: <br />
<br />
The mistakes is on 2421-Thaumatin-4, so there are two PCR needed to fix the mistake. <br />
<br />
The first one use primers 2421-Thaumatin-1 & 4 <br />
<br />
The second one use primers 2421-Thaumatin-5 & 18 <br />
<br />
== '''http://52.1.104.161/eln/images/3/36/2421_PCR.JPG''' ==<br />
<br />
<br />
<br />
<br />
<br />
== '''Result intepretation/Summary''' ==<br />
<br />
== '''Conclusion''' ==<br />
<br />
== '''Result intepretation/Summary''' ==<br />
<br />
== '''Conclusion''' ==<br />
[[Category:Pages with broken file links]] [[Category:Cloning]]</div>Excellgen