Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2016/02/10

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Overview

DBN012 (HA1:HPK:CFP:CFP:EM7:ZeoR:HA2) ligation results:

  • negative control (DBN011 E/X only): 4 colonies
  • treatment (HA1 E/S & DBN011 E/X): ~40 colonies

Going to pick 4 colonies and start liquid cultures.

DNA Quantification

Plasmid concentrations are all between 60-80 ng/µL

Gel Electrophoresis

Digested with NotI at 37 °C for 10 minutes, then ran on the gel at 100 V for 60 minutes. Expect a single band at 5875 bp (no NotI cut site in HA1 part).

Gel results:

File:2016-02-11 DBN012 NotI digest.tif

Colonies 2 and 3 look good, 1 is hard to tell but looks okay, 4 is not good. Going to proceed with sequencing colonies 2 and 3, using both biobrick forward and reverse primers.

Sample ID Template conc. (ng/µL) Template vol. (µL) Primer Primer vol. (µL) H2O (µL)
DBN012 2 fwd 60 1 P0001 2 3
DBN012 2 rev 60 1 P0002 2 3
DBN012 3 fwd 63 3 P0001 2 1
DBN012 3 rev 63 3 P0002 2 1