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

From OpenWetWare

Jump to: navigation, search
Today's project is... Main project page
Previous entry      

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:

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 fwd601P000123
DBN012 2 rev601P000223
DBN012 3 fwd633P000121
DBN012 3 rev633P000221


Personal tools