Hello to everyone,
I'm trying to amplify nuclear markers from old museum specimens of beetle (not very old, sampled 10-15 years ago) , I thought on the Wingless fragment, it's obviously a hard work an I don't expect to see much bands in the gels, anyway, anyone have suggestion about how to set the PCR for this goal?
Have you tried to amplify these yet or just trying to get started?
The wingless fragment can be amplified using a pretty standard PCR (depending on your primers) A standard one that colleagues use is:
94C - 3min (hotstart)
[ 94C-30s, 49-51C- 30s, 72C-1min] x36 cycles
72C - 10min for final extension.
The major factor in this will be how well your insect specimen was preserved, i.e. how degraded your DNA is. I've had well-preserved specimens (collected in EtOH and dry pinned) up to 20years old amplify just fine, while others only 5 years old not work for anything. You could try other types of cycles, like a touchdown reaction or load extra template and play with MgCl2 concentrations to help. If you get a faint band, you could also do a nested PCR, increase the number of cycles, or simply throw the samples back on the thermocycler for an extra 5 cycles.
Have you extracted these yet? How were you going to do this? There are a couple small tricks for certain methods that can maximize DNA yields from small or museum preserved specimens.
Thank you for your answers!
I already extracted some mtDNA from those samples, but I think that the nuclear is not well preserved. I did several tries without results.
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