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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

in midi culture 8/11repeat seq. w/ alt. prot.in seq 8/10in seq 8/10replated 8/11
3TCS3 9TCS3 15TCS3
     
 6TCS69TCS612TCS6 
     
3TCS96TCS99TCS912TCS915TCS9
 

 

in

seq #2 8/

11

in mini culture 8/11

(also replated)

repeat seq. w/ alt. prot.

26

 

plated 8/26replated 8/11
 6TCS129TCS1212TCS12 
 in seq 8/10in seq 8/11

 

 

 

 
3TCS15 9TCS15 15TCS15
    in seq