User:Stuart McKellar/Notebook/Bird Sex Testing/2012/12/03: Difference between revisions
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*B:2SWWBD | *B:2SWWBD | ||
*D:R5#51 | *D:R5#51 | ||
==60°C== | |||
Aim - try to see if results are better at 60°C | |||
method: | |||
Follwing exact same protocol as here: | Follwing exact same protocol as here: | ||
[http://openwetware.org/wiki/User:Stuart_McKellar/Notebook/Bird_Sex_Testing/2012/12/02]( | [http://openwetware.org/wiki/User:Stuart_McKellar/Notebook/Bird_Sex_Testing/2012/12/02] | ||
Master mix for all 8 samples plus -ve control(10 units of MM): | |||
*25ul buffer (10x) | |||
*20ul dNTPs | |||
*15ul MgCl2 | |||
*1ul Taq | |||
*25ul P2 primer (F) | |||
*25ul P8 primer (R) | |||
*129ul H<sub>2</sub>O | |||
Tubes labelled as follows: | |||
*AX:1SWWBD | |||
*BX:2SWWBD | |||
*CX:R5#9 | |||
*DX:R5#51 | |||
*EX:R5#52 | |||
*FX:R5#100 - (i htink i missed this one) | |||
*GX:Pink Gold | |||
*HX:banjo | |||
*-ve: 1ul water | |||
PCR the same except annealing temp is 60°C (user 77 file 81). | |||
results to be determined via agar gel E.Phoresis | |||
==55C== | |||
Same as above, samples AT,BT..etc | |||
PCR anneal at 55°C | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | ||
Revision as of 00:53, 3 December 2012
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Visualising lori's p2/p8 on 1% gelAim: differentiate between male and female loris using PCR product of p2/p8 primers and running on 1% gel. Method: Made up a 1% gel using the life technologies SYBR safe green dye. No dye was added, as the soln was already a 1:1 dilution of dye in TBE buffer. It was easy to make up and dissolved the agar readily. Results: Gels can be seen here: [1] It looks as though the samples A and B are male and that D is female, with the rest being ambiguous.
60°CAim - try to see if results are better at 60°C method: Follwing exact same protocol as here: [2] Master mix for all 8 samples plus -ve control(10 units of MM):
Tubes labelled as follows:
PCR the same except annealing temp is 60°C (user 77 file 81). results to be determined via agar gel E.Phoresis 55CSame as above, samples AT,BT..etc PCR anneal at 55°C | |
