User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/14: Difference between revisions

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* Do PCR for all fragments with Phusion polymerase, use fresh dNTP's (from Rene)
* Do PCR for all fragments with Phusion polymerase, use fresh dNTP's (from Rene)


* KAH
# '''Gal4DB-mCh''' (1170 bp): Template - KAH228 plasmid, F - Gal4DB f1 (Tm = 47), R - mCh r1 (Tm = 56)
*
# '''ATF2''' (906 bp): Template - iPSC cDNA (Brafman), F - ATF2_f1 (Tm = 56) , R - ATF2_r1 (Tm = 58)
# '''MV10''' (5191 bp): Template - MV10 plasmid, F - MV10 f1 (Tm = 64), R - MV10 r1 (Tm = 69)


* '''MV10''' (5191 bp): Template - MV10 plasmid, F - MV10 f1, R - MV10 r1


* Gradient PCR to enhance 5 kb band
* MV10 - Gradient PCR to enhance 5 kb band
# 68°C
# 68°C
# 67.3°C
# 67.3°C
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Conclusions
Conclusions
* tba
* tba
* Gal4DB-mCh and ATF2




Revision as of 09:43, 14 April 2015

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04/14/15

  • LCR Development


LCR Protocol Development

  • Refer to entry 04/06/2015
  • Do PCR for all fragments with Phusion polymerase, use fresh dNTP's (from Rene)
  1. Gal4DB-mCh (1170 bp): Template - KAH228 plasmid, F - Gal4DB f1 (Tm = 47), R - mCh r1 (Tm = 56)
  2. ATF2 (906 bp): Template - iPSC cDNA (Brafman), F - ATF2_f1 (Tm = 56) , R - ATF2_r1 (Tm = 58)
  3. MV10 (5191 bp): Template - MV10 plasmid, F - MV10 f1 (Tm = 64), R - MV10 r1 (Tm = 69)


  • MV10 - Gradient PCR to enhance 5 kb band
  1. 68°C
  2. 67.3°C
  3. 65.9°C
  4. 63.9°C
  5. 61.4°C
  6. 59.6°C
  7. 58.1°C
  8. 57°C
Reagent Rxn1-8 (x9) Expected:
1. Gal4DB-mCh = 1170
3,4. ATF2 = 906
5,6. MV10 = 5191 		Hover name
10 μL/lane, 1% agarose; Ladder
Template 0.2 1.8
10 uM fwd primer 1.0 9.0
10 uM rev primer 1.0 9.0
10 mM dNTPs 1.0 9.0
Phusion pol. 0.5 4.5
5x HF buffer 5.0 45.0
dH2O 41.3 371.7
  50.0

Program: Phusion (block A) - edited to include 65°C - 55°C annealing gradient

  • 98°C, 3 min
  • 30x[98°C, 10 sec; 68°C - 57°C, 30 sec; 72°C, 2.5 min]
  • 72°C, 10 min
  • 4°C ∞


Conclusions

  • tba


  • Gal4DB-mCh and ATF2






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