Ob/ob Genotyping

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