User:Stuart McKellar/Notebook/Bird Sex Testing/2012/11/23

From OpenWetWare
Jump to navigationJump to search
The printable version is no longer supported and may have rendering errors. Please update your browser bookmarks and please use the default browser print function instead.
Project name <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>

PCR optimisation

Yesterday failed to yield a single successful result. The two steps that were universal across all samples were number of cycles, an extended 72C period of 20 mins after 25 cycles, and 0.5U taq.

Aim: Today I will try with 3 different levels of taq and dna and run everything else the same as for the initial successful run. The tubes contents will be as follows:

  • 1 x Master Mix(uL):
  • 2.5 Buffer (10x)
  • 2.5 Forward (P2)
  • 2.5 Reverse (P8)
  • 2.5 dNTPs
  • 1.5 MgCl2
  • 10 H20

DNA From R5#9 (DNA 186.53 ng/ul)

Then as follows for each tube:

  • AA: 0.5ul DNA, 0.5U Taq; H20= 2.5
  • AB: 0.5ul DNA, 1.0U Taq; H20= 2.0
  • AC: 0.5ul DNA, 1.5U Taq; H20= 1.5
  • AD: 1.0ul DNA, 0.5U Taq; H20= 2.0
  • AE: 1.0ul DNA, 1.0U Taq; H20= 1.5
  • AF: 1.0ul DNA, 1.5U Taq; H20= 1.0
  • AG: 2.0ul DNA, 0.5U Taq; H20= 1.0
  • AH: 2.0ul DNA, 1.0U Taq; H20= 0.5
  • AI: 2.0ul DNA, 1.5U Taq; H20= 0.0
  • A-ve: 0.0ul DNA 1.0U Taq; H20= 2.5

All of the tubes had 24.5 ul total. They had 21ul of MM, instead of 21.5.


Visualised on a 1% gel. 500V, 75mA for about 30 minutes. Bars everywhere! Sample looks female.



Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Stuart_McKellar/Notebook/Bird_Sex_Testing/2012/11/23&oldid=658880"