Editing 2421-pEC-Thaumatin

== '''Cloning of <span dir="auto">2421-pEC-Thaumatin</span>''' ==
<div>'''Date:&nbsp;''' 2015-4<br/></div>
'''By: Xin'''

'''Results:'''

Gene Synthesis: about 750bp. The vector PEC (1454)

'''[[File:2421, 2418, 2434 clone 4-16-15.JPG|313x151px|RTENOTITLE]]'''

==  ==

&nbsp; Digested by NdeI /KpnI, ligation, transformation.

== '''Screening:''' ==

ini-preparation

[[File:2421screening.JPG|RTENOTITLE]]

==  ==

Send #2 to sequence on April 23,2015

Send #3 to sequence on April 25, 2015

'''''Fix''''' #3 on April 26, 2015:

use #3 sequenced on (April 25, 2015) as template,

PCR fragment I: the oligos 2421-Thaumatin-1 and 2421-Thaumatin-4

==  ==

PCR fragment II: the oligos 2421-Thaumatin-5 and 2421-Thaumatin-18

[[File:2423,2436,2421 PCR.JPG|343x143px|RTENOTITLE]]

Join two fragments together.

[[File:2436, 2423, 2421 PCR.JPG|416x155px|RTENOTITLE]]

==  ==

'''Screening''':

[[File:1916 2423 2421 screening.JPG|475x152px|RTENOTITLE]]

&nbsp;1,2,3 and 4 colonies are positive.

==  ==

Send #4 to sequence on 5/5/2015, the sequencing is still same as the template.

== ''Fix 5/5/2015'' ==

The template used#2&nbsp; (sequenced on April 23,2015)

The mistakes was happened on oligo 2421-Thaumatin-1 / -2&nbsp;

There are two ways to fix:

One is to use 2421-Thaumatin-1 and -18 redo a PCR.

==  ==

[[File:2412.JPG|391x135px|RTENOTITLE]]

The second way to fix is to do a PCR use 2421-Thaumatin-3 and -18.

[[File:2421;1916.JPG|213x189px|RTENOTITLE]]

==  ==

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.

[[File:1916,2421.JPG|193x144px|RTENOTITLE]]

==  ==

=== '''Screening''': ===

screening 4 colonies first (from Fix method one plate)

[[File:2421 5-10-15.JPG|331x201px|RTENOTITLE]]

==  ==

Send #4 to sequence, the result is still same as previous.

&nbsp;Rescreening 8 colonies, 1-4 are the colonies conducted by fix method one; 5-8 are the colonies conducted by fix method two.

[[File:2421,2412.JPG|402x181px|RTENOTITLE]]

==  ==

Send sequencing #6 to sequencing. There is still one mistake, which is different as previous mistakes.&nbsp;

Rescreen 7 colonies (from fix method II plate).

&nbsp;[[File:2421 screening 5-15-15.JPG|296x157px|RTENOTITLE]]

Send #2 for sequencing on 5/15/2015.

== ''Fix on 5/28/2015'' ==

Use the sequencing on 5-15-2015 as template: 

The mistakes is on 2421-Thaumatin-4, so there are two PCR needed to fix the mistake. 

The first one use primers 2421-Thaumatin-1 & 4 

The second one use primers 2421-Thaumatin-5 & 18 

== '''http://52.1.104.161/eln/images/3/36/2421_PCR.JPG''' ==





== '''Result intepretation/Summary''' ==

== '''Conclusion''' ==

== '''Result intepretation/Summary''' ==

== '''Conclusion''' ==
[[Category:Pages with broken file links]] [[Category:Cloning]]