Haynes Lab:Notebook/CRISPR Editing/2014/09/17: Difference between revisions

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(Autocreate 2014/09/17 Entry for Haynes_Lab:Notebook/CRISPR_Editing)
 
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==07//2014==
==09/17/2014==


Added 1ul Enhancer and 1ul Surveyor nuclease on ice to the annealing reactions from yesterday. Incubate for 60 min at 42 in thermal cycler. Ran on 1% gel:


<br><br>
Trying to increase sample genomic DNA PCR product concentration. Varied starting template concentration and reaction volume:
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Rxn #'''
| align="center" style="background:#f0f0f0;"|'''template'''
| align="center" style="background:#f0f0f0;"|'''uL water'''
| align="center" style="background:#f0f0f0;"|'''5x phusion HF buffer'''
| align="center" style="background:#f0f0f0;"|'''10mM DNTPs'''
| align="center" style="background:#f0f0f0;"|'''10uM f primer'''
| align="center" style="background:#f0f0f0;"|'''10uM r primer'''
| align="center" style="background:#f0f0f0;"|'''Template'''
| align="center" style="background:#f0f0f0;"|'''DMSO'''
| align="center" style="background:#f0f0f0;"|'''Phusion'''
| align="center" style="background:#f0f0f0;"|'''Total rxn V'''
|-
| 1||gRNA 20 +dox rep 1||10.8||4||0.4||1||1||2.00||0.6||0.2||20.0
|-
| 2||gRNA 20 +dox rep 1||30.0||10||1||2.5||2.5||2.00||1.5||0.5||50.0
|-
| 3||gRNA 20 +dox rep 1||8.8||4||0.4||1||1||4.00||0.6||0.2||20.0
|-
| 4||gRNA 20 +dox rep 1||28.0||10||1||2.5||2.5||4.00||1.5||0.5||50.0
|}


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Revision as of 09:05, 17 September 2014

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09/17/2014

Added 1ul Enhancer and 1ul Surveyor nuclease on ice to the annealing reactions from yesterday. Incubate for 60 min at 42 in thermal cycler. Ran on 1% gel:



Trying to increase sample genomic DNA PCR product concentration. Varied starting template concentration and reaction volume:

Rxn # template uL water 5x phusion HF buffer 10mM DNTPs 10uM f primer 10uM r primer Template DMSO Phusion Total rxn V
1 gRNA 20 +dox rep 1 10.8 4 0.4 1 1 2.00 0.6 0.2 20.0
2 gRNA 20 +dox rep 1 30.0 10 1 2.5 2.5 2.00 1.5 0.5 50.0
3 gRNA 20 +dox rep 1 8.8 4 0.4 1 1 4.00 0.6 0.2 20.0
4 gRNA 20 +dox rep 1 28.0 10 1 2.5 2.5 4.00 1.5 0.5 50.0


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