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hEF1a:CD79B-pTet:mRFP-Gal4VP16

PCR: when we redesigned primers to fix dimerization issues, we accidentally introduced a missense mutation in the third codon from the end of CD79B. We decided to use SDM to fix the mutation instead of rePCRing

06/19

  • SDM

06/23

  • Gel verification of SDM --> no bands: possible that there wasn't enough amplification. will run SDM again and transform product directly 

06/24

  • SDM and DpnI digest 
  • transform 

06/25

  • No colonies --> could be because a small amount of the digest was transformed.
  • all of digest transformed

06/26

  • No colonies

hEF1a:CD79B-XnTCSXc-Gal4VP16

3TCS3 9TCS3 15TCS3
in midi culture
  
in midi culture
  
waiting to be midi'ed
 
 6TCS69TCS612TCS6 
 
in midi culturein midi culture
   
in midi culture
 
3TCS96TCS99TCS912TCS915TCS9
in midi culturesend for seq.replaterepeat seq.miniprep
 

 

seq #2 8/26

 

plated 8/26
 6TCS129TCS1212TCS12 
 
miniprepsend for seq.

 

 

 

repeat seq.

 
3TCS15 9TCS15 15TCS15
miniprep
  
rertransform?
  
restreak
in seq