User:Stuart McKellar/Notebook/Bird Sex Testing/2012/12/03
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(Autocreate 2012/12/03 Entry for User:Stuart_McKellar/Notebook/Bird_Sex_Testing) |
Current revision (03:53, 3 December 2012) (view source) (→Visualising lori's p2/p8 on 1% gel) |
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| - | == | + | ==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: [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 | ||
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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 | |
