Excellgen at 20:31, 18 May 2015

2015-05-18T20:31:35Z

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== '''Cloning of 2412 pEC2-AaeMutS''' ==

'''Purpose:''' Cloning of 2412 pEC2-AaeMutS

'''Date: '''2015 March, April ''' ''

'''''Clone ID: 2412'''''

''Experimenters:'''Xu Xin'''''

== '''Results''' ==

== ==

=== cloning ===

Fix: 2412 sequecing: use the below primers to do 7 PCR, 

1-1 + 1-12; 1-13 + 1-16; 1-17 + 1-18; 1-19 + 2-14; 2-15 + 3-2 ; 3-3 + 3-6; 3-7 + 3-22

then 1-1 + 3-22

== ==

[[File:2412 fix PCR Fragments 1-7 3-19-14.JPG|240x144px|RTENOTITLE]]

Cut gel for the 7 fragments, put together for gel purification. 

Use Olig for 1-1 and 3-22 for a big fragment PCR (annealing tem: 58, with should be 2500 bp, use 2x primer master mix).

== ==

[[File:2412-big fragment PCR.JPG|149x202px|RTENOTITLE]]

 The PCR product was purified from gel, and digestion with vector 1454 by Nde1 and XhoI; ligation, transformation and screening.

== '''[[File:2412-fix--.JPG|259x102px|RTENOTITLE]]''' ==

'''The PCR screening only use 25 cycles, the band is hard to see.'''

'''But on the gel (in the dark), the colony 7, 8, 9 show a band clearly.'''

'''incolulation 7, 8, 9 and mini plasmid prepation, digested by XhoI + NdeI'''.

[[File:2412-fix-screening-3-27-15.jPG|149x131px|RTENOTITLE]]

digested again with orginal 2412 plasmid to compare with

== ==

[[File:2412 screen 3-30-15.JPG|163x194px|RTENOTITLE]]

Repeat again on April 18, 2015,

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

Digest by NdeI/XhoI and didn't find the expected cuting size, which could be caused the cutting size is too big (2500 bp).

[[File:2412,1916 screeening 4-21-15 .JPG|333x150px|RTENOTITLE]]

Redigested by BamHI and NdeI, with 2412 positive control.The clonies# 2 and 6 have the expected product. 

 [[File:4-21-15 2412 screeningetc.jpg|450x178px|RTENOTITLE]]

The next step is sequencing.

== '''''Fix'''''on April 28, 2015 ==

The clony #6 was sequencd, there were still a few mistakes, and will be fixed.

{| style="width: 392px" cellpadding="1" cellspacing="1" width="500" border="1"
|-
| style="width: 83px" |   Fragments 
| style="width: 241px" |      PCR Primers 
| style="width: 51px" |   Size 
|-
| style="width: 83px" |    1 
| style="width: 241px" | 2412-AaeMutS-1-1; 1-14    
| style="width: 51px" | 600bp 
|-
| style="width: 83px" |    2 
| style="width: 241px" | 2412-AaeMutS-1-17; 2-20  
| style="width: 51px" | 1021bp 
|-
| style="width: 83px" |    3 
| style="width: 241px" | 2412-AaeMutS-1-1; 1-14   
| style="width: 51px" | 820bp 
|}

[[File:2412 PCR fragments.JPG|RTENOTITLE]]

Join the three fragments together, using the purified PCR product,

10umol 2412-AaeMutS-1-1,

10umol 2412-AaeMutS-1-14,

1umol 2412-AaeMutS-3-1,

1umol 2412-AaeMutS-3-2,

1umol 2412-AaeMutS-3-3,

1umol 2412-AaeMutS-3-22.

[[File:2412 big fragment.JPG|RTENOTITLE]]

2500bp PCR product was what we interested. Gel purified, digestion, ligation (5/7/2015)

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

[[File:2412;21916.JPG|RTENOTITLE]] ===

'''''Screening'''''

#9 colony is the positive control

[[File:2412;21916.JPG|RTENOTITLE]]

The colonies# 4,5,6 and 8 are positive. #4 was sent to sequencing on 5/10/15.

'''PCR again:'''

During experiment, the concentration (ng/ul) for oligos on plate 2-9-2015 were much lower than other oligo.

More oligos (on the plate 2-9-2015) were added into the PCR reaction, and carried PCR again.

During experiment, the concentration (ng/ul) for oligos on plate 2-9-2015 were much lower than other oligo.

More oligos (on the plate 2-9-2015) were added into the PCR reaction, and carried PCR again.

''[[File:2412.JPG|550x173px|RTENOTITLE]]''

digestion, ligation, transformation.

Screening

[[File:2421 2412 screening.JPG|477x188px|RTENOTITLE]]

3 is the positive colony. But this one didn't send to sequencing until the the sequencing result#4 come back and too many mistakes.

== '''''Fix on 5/18/2015''''' ==

Use the #4 sequenced on 5/10/2015 as template to fix again.

There are three sections PCR was conducted:

{| cellpadding="1" cellspacing="1" width="205" border="1"
|-
| style="width: 43px" | 1-1 
| style="width: 71px" |   1-16 
| style="width: 75px" |   700bp 
|-
| style="width: 43px" | 1-17 
| style="width: 71px" |    3-2 
| style="width: 75px" |  1000bp 
|-
| style="width: 43px" | 3-3 
| style="width: 71px" |   3-22 
| style="width: 75px" |  780bp 
|}

[[File:2412 PCR.JPG|150x143px|RTENOTITLE]]

Combined all the PCR together as a template to carried a big PCR.

2412 pEC2-AaeMutS - Revision history

Excellgen at 20:31, 18 May 2015