Haynes Lab:Notebook/CRISPR Editing/2014/10/07

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10/07/2014

Ran a gel of some of the samples from the qPCR plate to check for primer dimer products

700

Weird things about this gel:

Bands in my no template control lanes
Bands are fuzzy like it's not a single product
Bands look too big
Bands with the same primer set seem to be different sizes


Possibility 1: I made a mistake and added template to my no template controls

Counter: The likely way I would do this would be to add a correctly diluted sample but the Cp of these wells is later than my lowest dilution on my curve
Counter: Doesn't explain bands of different sizes for same primer set

Possibility 2: I'm only measuring primer dimer

Counter: Why are my dilution curves so perfect? Seems the Cp is dependent on template concentration.



Sent PCR products for Luc14 gDNA Surveyor PCR products for each gRNA out for sequencing

Sample Primer ' F/R Distance from gRNA
Luc14 gRNA 20 Srvyr PCR P165 gRNA 021 US P F F 149
Luc14 gRNA 21 Srvyr PCR P164 gRNA 020 US P R R 166
Luc14 gRNA 22 Srvyr PCR P165 gRNA 021 US P F F 103
Luc14 gRNA 27 Srvyr PCR P155 gRNA 026 ES P F F 121
Luc14 gRNA 29 Srvyr PCR P162 gRNA 030 ES P R R 125
Luc14 Srvyr PCR P165 gRNA 021 US P F F 149
Luc14 Srvyr PCR P164 gRNA 020 US P R R 166
Luc14 Srvyr PCR P155 gRNA 026 ES P F F 121
Luc14 Srvyr PCR P162 gRNA 030 ES P R R 125