Haynes Lab:Notebook/CRISPR Editing/2014/07/14: Difference between revisions
Rene M Davis (talk | contribs) (Autocreate 2014/07/14 Entry for Haynes_Lab:Notebook/CRISPR_Editing) |
Rene M Davis (talk | contribs) |
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==07//2014== | ==07/14/2014== | ||
Ligation of the other gRNAs. | |||
<br><br>'''Forgot backbone control but I've been getting crazy high background anyway so it wouldn't change my behavior'''<br><br> | |||
gRNA 13, 1T, 1B, 3T, 3B<br> | |||
'''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. | |||
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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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Plated a second 6-well plate for transfections tomorrow. | |||
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07/14/2014Ligation of the other gRNAs.
gRNA 13, 1T, 1B, 3T, 3B 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.
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