Haynes Lab:Notebook/CRISPR Editing/2014/07/14

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(Autocreate 2014/07/14 Entry for Haynes_Lab:Notebook/CRISPR_Editing)
Current revision (16:44, 15 July 2014) (view source)
(07//2014)
 
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Ligation of the other gRNAs.
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<br><br>'''Forgot backbone control but I've been getting crazy high background anyway so it wouldn't change my behavior'''<br><br>
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gRNA 13, 1T, 1B, 3T, 3B<br>
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'''may have contaminated 1T ligation reaction with some of 13 ligation reaction. '''
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Followed the protocol exactly.
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http://www.genome-engineering.org/crispr/wp-content/uploads/2014/05/CRISPR-Reagent-Description-Rev20140509.pdf
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Transformed 2μL of ligation reaction into 60μL of DH5αT competent cells. Long transformation, 950μL SOC.
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<br><br>
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Plated a 6-well plate to retry the G418 selection. Triple checked that I did the math correctly and that my final concentrations are correct and consistent with literature. Found several sources that select at 800μg/mL. Added G418 to final concentrations of 0, 500, 800, 1000, 1500, 2000μg/mL.
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<br><br>
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Plated a second 6-well plate for transfections tomorrow.
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07/14/2014

Ligation of the other gRNAs.

Forgot backbone control but I've been getting crazy high background anyway so it wouldn't change my behavior

gRNA 13, 1T, 1B, 3T, 3B
may have contaminated 1T ligation reaction with some of 13 ligation reaction.

Followed the protocol exactly. http://www.genome-engineering.org/crispr/wp-content/uploads/2014/05/CRISPR-Reagent-Description-Rev20140509.pdf

Transformed 2μL of ligation reaction into 60μL of DH5αT competent cells. Long transformation, 950μL SOC.




Plated a 6-well plate to retry the G418 selection. Triple checked that I did the math correctly and that my final concentrations are correct and consistent with literature. Found several sources that select at 800μg/mL. Added G418 to final concentrations of 0, 500, 800, 1000, 1500, 2000μg/mL.



Plated a second 6-well plate for transfections tomorrow.


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