Team:SUSTC-Shenzhen/Notebook/CRISPR/Successful-fill-in-plasmid-(G56-1)-amplification

From 2014.igem.org

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       name=Verifications for ‘struggles for our second- time Fill-in’ with EcoRI, MfeI|
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       name=Successful fill-in plasmid (G56-1) amplification|
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       date=2014/8/14|
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       date=2014/8/18|
       goal=}}
       goal=}}
==Materials==
==Materials==
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Enzymes: EcoRI & MfeI    Buffer: Buffer 2.1
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SOC medium  400μl  <br>
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Amp Agar plates  2* plates  <br>
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Inoculating loop <br>
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Successful fill-in plasmid:PB5 - HS4 - 5xUAS - mCherrynuc - 2A - Bla - loxN - GpA - HS4 - PB3<br>
==Methods==
==Methods==
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=== Digestion system：(ul) ===
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1.Place the tubes on ice, allow cell to thaw on ice. <br>
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2.Divide the competent cells into 2 tubes,50μL each tube, Mark them with G1, G2 .<br>
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{|class="table"
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3.Add 2μL plasmid to each tube, shaking slightly, keep the bacteria in ice at 4°C for 45min.<br>
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!MfeI & EcoRI
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4.Put the samples at 42°C for 90s to transfer the plasmids into the cells.<br>
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!Total volume
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5.Immediately transfer the samples to ice. And keep the samples in ice for 2 min.<br>
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!DNA
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6.Transfer each one of the samples to 0.2 ml of liquid SOC media for 45 min at 37°C，200 rpm shaker.<br>
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!Buffer 2.1
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7.Centrifuge the samples 4000 rpm, 5min. <br>
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!MfeI
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8.Discard the supernatant liquid.Add 50μL samples to amp LB agar plant respectively and distribute the bacteria in plate.<br>
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!EcoRI
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9.Incubate the plates at 37°C .<br>
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!ddH2O
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|-
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|G (1) 689ng/ul
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|10
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|2.2
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|1
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|1.5
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|1.5
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|3.8
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|-
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|G (2) 754ng/ul
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|10
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|1.9
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|1
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|1.5
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|1.5
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|4.1
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-
|}
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{|class="table"
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!MfeI / EcoRI
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!Total volume
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!DNA
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!Buffer 2.1
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!MfeI / EcoRI
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!ddH2O
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-
|-
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|G (1) 689ng/ul
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|10
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|2.2
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|1
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|1.5
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|5.3
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-
|-
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|G (2) 754ng/ul
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|10
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|1.9
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|1
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|1.5
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|5.6
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|}
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Incubate at 37°C for 4hours
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=== Results of electrophoresis===
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{{SUSTC-Image|wiki/images/7/7c/SUSTC-Shenzhen-Verifications-for-succeed-Fill-in-plasmid.jpg}}
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The bands of G1 were to compliated to analyze.  
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For G2, according to our assumption, If the EcoRI site had been filled out, the profile of its bands should be congruent to that of the original plasmid; if not, it should be aligned to that of the M digestion. From the picture, we can see that except for the abnormal bands of double digestion (EcoRI and MfeI), the other DNA bands along single digestion tracks could roughly imply that the EcoRI site had been removed from the plasmid by Fill-in. Because the bands of EcoRI digestion were similar to those of the original plasmid which hadn’t been digested by any restriction enzymes. While the very band of MfeI digestion was obviously behind those of E digestion and original plasmid, indicating that digestion with MfeI had linearized the plasmid.  
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Revision as of 07:21, 17 October 2014

Team SUSTC-Shenzhen

Notebook

Elements of the endeavor.

Contents


Successful fill-in plasmid (G56-1) amplification

2014/8/18


Materials

SOC medium 400μl
Amp Agar plates 2* plates
Inoculating loop
Successful fill-in plasmid:PB5 - HS4 - 5xUAS - mCherrynuc - 2A - Bla - loxN - GpA - HS4 - PB3

Methods

1.Place the tubes on ice, allow cell to thaw on ice.
2.Divide the competent cells into 2 tubes,50μL each tube, Mark them with G1, G2 .
3.Add 2μL plasmid to each tube, shaking slightly, keep the bacteria in ice at 4°C for 45min.
4.Put the samples at 42°C for 90s to transfer the plasmids into the cells.
5.Immediately transfer the samples to ice. And keep the samples in ice for 2 min.
6.Transfer each one of the samples to 0.2 ml of liquid SOC media for 45 min at 37°C，200 rpm shaker.
7.Centrifuge the samples 4000 rpm, 5min.
8.Discard the supernatant liquid.Add 50μL samples to amp LB agar plant respectively and distribute the bacteria in plate.
9.Incubate the plates at 37°C .

Maintained by the iGEM team SUSTC-Shenzhen.

Licensed under CC BY 4.0.

  • ©2014 SUSTC-Shenzhen

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