User:Stuart McKellar/Notebook/Bird Sex Testing/2012/12/03

From OpenWetWare

< User:Stuart McKellar | Notebook | Bird Sex Testing | 2012 | 12(Difference between revisions)
Jump to: navigation, search
(Autocreate 2012/12/03 Entry for User:Stuart_McKellar/Notebook/Bird_Sex_Testing)
Current revision (02:53, 3 December 2012) (view source)
(Visualising lori's p2/p8 on 1% gel)
 
(2 intermediate revisions not shown.)
Line 6: Line 6:
| colspan="2"|
| colspan="2"|
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
-
==Entry title==
+
==Visualising lori's p2/p8 on 1% gel==
-
* Insert content here...
+
Aim: 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: [http://imgur.com/a/5omku]
 +
 +
It looks as though the samples A and B are male and that D is female, with the rest being ambiguous.
 +
*A:1SWWBD
 +
*B:2SWWBD
 +
*D:R5#51
 +
 +
==60°C==
 +
 +
Aim - try to see if results are better at 60°C
 +
 +
method:
 +
 +
Follwing exact same protocol as here:
 +
[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. ##### -->

Current revision

Project name Main project page
Previous entry      Next entry

Visualising lori's p2/p8 on 1% gel

Aim: 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.

  • A:1SWWBD
  • B:2SWWBD
  • D:R5#51

60°C

Aim - 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):

  • 25ul buffer (10x)
  • 20ul dNTPs
  • 15ul MgCl2
  • 1ul Taq
  • 25ul P2 primer (F)
  • 25ul P8 primer (R)
  • 129ul H2O

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


Personal tools