Team:SUSTC-Shenzhen/Notebook/CRISPR/Verification-for-pX330-mCherry-pX330-mCherry-GAL4-enzyme-digestion

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{{:Team:SUSTC-Shenzhen/templates/page-header|
       title=Notebook|
       subtitle=Elements of the endeavor.}}

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{{:Team:SUSTC-Shenzhen/templates/notebook-main|
       name=Verification for pX330+mCherry, pX330+mCherry+GAL4 by enzyme digestion|
       date=2014/8/22|
       goal=}}

==Materials==

Enzymer: BbsI, AgeI, EcoRI
Buffe: Buffer2.1, Cutsmart Buffer

==Methods==

===Digestion system===

BbsI digestion system:

{|class="table"
!
!Component
!Volume
|-
|BbsI
|0.5ul
|-
|DNA
|0.5ul
|-
|10X NEBuffer 2.1
|1ul
|-
|ddH2O
|8ul
|-
|Total
|10ul
|}

EcoRI digestion system:

{|class="table"
!
!Component
!Volume
|-
|EcoRI 
|0.5ul
|-
|DNA
|0.5ul
|-
|10X NEBuffer 2.1
|1ul
|-
|ddH2O
|8ul
|-
|Total
|10ul
|}

AgeI digestion system:

{|class="table"
!
!Component
!Volume
|-
|AgeI 
|0.5ul
|-
|DNA
|0.5ul
|-
|10X NEBuffer 2.1
|1ul
|-
|ddH2O
|8ul
|-
|Total
|10ul
|}

BbsI and EcoRI digestion system:

{|class="table"
!
!Component
!Volume
|-
|BbsI 
|0.5ul
|-
|EcoRI  
|0.5ul
|-
|DNA
|0.5ul
|-
|10X NEBuffer 2.1
|1ul
|-
|ddH2O
|7.5ul
|-
|Total
|10ul
|}

AgeI and EcoRI digestion system:

{|class="table"
!
!Component
!Volume
|-
|AgeI 
|0.5ul
|-
|EcoRI  
|0.5ul
|-
|DNA
|0.5ul
|-
|10X NEBuffer 2.1
|1ul
|-
|ddH2O
|7.5ul
|-
|Total
|10ul
|}

=== Incubate at 37C, 4hours [12:46~17:00]===
=== Gel electrophoresis===

Making the agarose gel (1%):

{|class="table"
!Component
!volume
|-
|TAE  
|40ml
|-
|Agarose
|0.4g
|-
|Gene Green
|1ul
|}

Running conditions: 130V, 30min

==Results==

{{SUSTC-Image|wiki/images/8/85/SUSTC-Shenzhen-Verification-for-pX330-mCherry-pX330-mCherry-GAL4-by-enzyme-digestion.jpg}}

From the picture, we can see that: <br>
The position of the bands generated by singly digestion with AgeI or EcoRI aligned with that of the 6000bp indicated by Marker. And when digested with AgeI and EcoRI simultaneously, there were two bands appearing on the track, one is around 5000bp, and the other is nearly 800bp, both of which implied the success of our ligation (point-mutated mCherry fragment was inserted into pX330 vector).<br>
What’s more, from the results of double digestion by BbsI and EcoRI as well as single digestions with BbsI or EcoRI, we can proof the successful mutation-out of BbsI site on mCherry fragment again. (a. Figure of double digestion by BbsI and EcoRI was the same as that of the single digestions with EcoRI; b. Figure of single digestions with BbsI was the same as that of the original plasmid without digestion.)<br>