User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/10: Difference between revisions

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==04/10/15==
==04/10/15==
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* LCR development - Backbone PCR, Phusion and temp gradient, re-do
* LCR development - Backbone PCR, Phusion and temp gradient, re-do (do this on Monday)




Revision as of 17:12, 10 April 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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04/10/15

  • LCR development - Backbone PCR, Phusion and temp gradient, re-do (do this on Monday)


LCR development - Backbone PCR

  • Repeat PCR from 04/09/15, use fresh dNTP's
  • MV10 (5191 bp): Template - MV10 plasmid, F - MV10 f1, R - MV10 r1
  • 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




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