Difference between revisions of "Ob/ob Genotyping"

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(added other gene specific primer)
(added nots on how to make primer mixes and added band sizes)
 
(One intermediate revision by the same user not shown)
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* 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/ob''' and is:
+
The PCR program is called '''ob-ob''' and is:
  
 
* 95 °C 2 min  
 
* 95 °C 2 min  
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==Bands==
 
==Bands==
[[File:ob_ob.jpg]]
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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)

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