User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/08

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04/08/15

  • LCR development - test 5'-phos primers for Gal4DB-mCh-ATF2_MV10 assembly



LCR - test 5'-phos primers

  1. Gal4DB-mCh (1170 bp): Template - KAH228 plasmid, F - Gal4DB f1, R - mCh r1
  2. ATF2 (906 bp): Template - iPSC cDNA (Brafman), F - ATF2_f1, R - ATF2_r1
  3. MV10 (5191 bp): Template - MV10 plasmid, F - MV10 f1, R - MV10 r1
Reagent Rxn1,2 Rxn3,4 Rxn5,6 Expected:
1,2. Gal4DB-mCh = 1170
3,4. ATF2 = 906
5,6. MV10 = 5191
Hover name
10 μL/lane, 1% agarose; Ladder
Template 0.2 2.0 2.0
10 uM fwd primer 1.0 1.0 1.0
10 uM rev primer 1.0 1.0 1.0
2x GoTaq green 12.5 12.5 12.5
dH2O 10.3 8.5 10.3
  25.0 25.0 25.0

Program: GOTAQ35cyc

  • 95°C, 3 min
  • 30x[95°C, 1 min; 57°C, 1 min; 72°C, 3 min]
  • 72°C, 3 min
  • 4°C ∞


Conclusions

  • Gal4DB-mCh seems correct, except for spurious bands. Purify this product and re-run on gel
  1. ATF2 is veyr faint. Retry PCR with more cycles, lower temp.
  2. MV10 uppermost band might be correct. Try Phusion and gradient PCR.



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