Editing 2412 pEC2-AaeMutS

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

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

'''Date:&nbsp;'''2015 March, April '''&nbsp;''

'''''<b>Clone ID: 2412</b>'''''

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

== '''Results''' ==

==  ==

=== cloning ===

<span style="font-family:arial,helvetica,sans-serif"><font style="font-size: 10pt" size="2">Fix:</font> 2412 sequecing: use the below primers to do 7 PCR,&nbsp;</span>

<span style="font-family:arial,helvetica,sans-serif"><font style="font-size: 10pt" size="2">1-1 + 1-12; 1-13 + 1-16</font>; <font style="font-size: 10pt" size="2">1-17 + 1-18</font>; <font style="font-size: 10pt" size="2">1-19 + 2-14</font>; <font style="font-size: 10pt" size="2">2-15 + 3-2</font>&nbsp;; <font style="font-size: 10pt" size="2">3-3 + 3-6</font>; <font style="font-size: 10pt" size="2">3-7 + 3-22</font></span>

<span style="font-family:arial,helvetica,sans-serif"><font style="font-size: 10pt" size="2">then 1-1 + 3-22</font></span>

==  ==

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

Cut gel for the 7 fragments, put together for gel purification.&nbsp;

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]]

&nbsp;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.&nbsp;





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

The next step is sequencing.

== '''''Fix'''''<span style="font-size:x-small">on April 28, 2015</span> ==

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" | &nbsp; Fragments<br/>
| style="width: 241px" | &nbsp;&nbsp;&nbsp;&nbsp; PCR Primers<br/>
| style="width: 51px" | &nbsp; Size<br/>
|-
| style="width: 83px" | &nbsp;&nbsp; 1<br/>
| style="width: 241px" | 2412-AaeMutS-1-1; 1-14 &nbsp;&nbsp;<br/>
| style="width: 51px" | 600bp<br/>
|-
| style="width: 83px" | &nbsp;&nbsp; 2<br/>
| style="width: 241px" | 2412-AaeMutS-1-17; 2-20&nbsp;<br/>
| style="width: 51px" | 1021bp<br/>
|-
| style="width: 83px" | &nbsp;&nbsp; 3<br/>
| style="width: 241px" | 2412-AaeMutS-1-1; 1-14 &nbsp;<br/>
| style="width: 51px" | 820bp<br/>
|}

[[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.

'''<span style="font-size:larger">PCR again:</span>'''

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<br/>
| style="width: 71px" | &nbsp; 1-16<br/>
| style="width: 75px" | &nbsp; 700bp<br/>
|-
| style="width: 43px" | 1-17<br/>
| style="width: 71px" | &nbsp;&nbsp; 3-2<br/>
| style="width: 75px" | &nbsp;1000bp<br/>
|-
| style="width: 43px" | 3-3<br/>
| style="width: 71px" | &nbsp; 3-22<br/>
| style="width: 75px" | &nbsp;780bp<br/>
|}



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

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