User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/09: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 55: Line 55:
* 72°C, 10 min
* 72°C, 10 min
* 4°C ∞
* 4°C ∞
Conclusions
* Poor amplification. Used stock dNTPs from old Harvard box.
* Repeat with fresh stocks dNTPs from Rene.




Revision as of 16:18, 10 April 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

04/09/15

  • LCR development - Backbone PCR, Phusion and temp gradient


LCR development - Backbone PCR

  • 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

  • Poor amplification. Used stock dNTPs from old Harvard box.
  • Repeat with fresh stocks dNTPs from Rene.




Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/04/09&oldid=879364"