Difference between revisions of "Ob/ob Genotyping"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (added other gene specific primer) |
Davebridges (Talk | contribs) (added nots on how to make primer mixes and added band sizes) |
||
| (One intermediate revision by the same user not shown) | |||
| Line 6: | Line 6: | ||
* ob/ob - WT-Lep-F: 5'- AAT GAC CTG GAG AAT CTC C -3' | * ob/ob - WT-Lep-F: 5'- AAT GAC CTG GAG AAT CTC C -3' | ||
* ob/ob - Lepob-R: 5'- GCA GAT GGA GGA GGT CTC A -3' | * ob/ob - Lepob-R: 5'- GCA GAT GGA GGA GGT CTC A -3' | ||
| + | |||
| + | ===Primer Mixes=== | ||
| + | Primers are resuspended at 100 uM, so add 3x10 uL of the primers to 970uL of water to make the primer mixes: | ||
| + | *For '''WT Primers''' combine both RFLP primers with the WT-Lep-F primer | ||
| + | *For '''Ob Primers''' combine both RFLP primers with the Lebob-R primer | ||
==PCR Reaction== | ==PCR Reaction== | ||
| − | The PCR program is called '''ob | + | The PCR program is called '''ob-ob''' and is: |
* 95 °C 2 min | * 95 °C 2 min | ||
| Line 18: | Line 23: | ||
==Bands== | ==Bands== | ||
| − | + | There should be a control band in all lanes at 191bp | |
| + | For the wt primers if there is one or more wt alleles there should be a band at 104bp | ||
| + | For the ob primers, if there is one or more ob alleles there should be a band at 123bp | ||
| + | |||
[[ Category:Genotyping ]] | [[ Category:Genotyping ]] | ||
Latest revision as of 19:59, 26 September 2013
Used method described in Ellet et al (http://dx.doi.org/10.1038/oby.2008.443)
Contents
Primers
- ob/ob RFLP-F: 5'- TGA GTT TGT CCA AGA TGG ACC -3'
- ob/ob RFLP-R: 5'- GCC ATC CAG GCT CTC TGG -3'
- ob/ob - WT-Lep-F: 5'- AAT GAC CTG GAG AAT CTC C -3'
- ob/ob - Lepob-R: 5'- GCA GAT GGA GGA GGT CTC A -3'
Primer Mixes
Primers are resuspended at 100 uM, so add 3x10 uL of the primers to 970uL of water to make the primer mixes:
- For WT Primers combine both RFLP primers with the WT-Lep-F primer
- For Ob Primers combine both RFLP primers with the Lebob-R primer
PCR Reaction
The PCR program is called ob-ob and is:
- 95 °C 2 min
- 40 cycles of:
- 95 °C 30 s
- 58 °C 30 s
- 72 °C 45 s
- 72 °C 7 min
Bands
There should be a control band in all lanes at 191bp For the wt primers if there is one or more wt alleles there should be a band at 104bp For the ob primers, if there is one or more ob alleles there should be a band at 123bp
