Behavioral Ecology Blog

Tuesday 9/18/07

Step 2 of the protocol calls for a restriction enzyme digest of genomic DNA. The idea is to chop up DNA into pieces small enough so that they can be easily cloned, but large enough to have a good chance of having a microsatellite AND enough flanking sequence to allow for primer development.

The authors of the protocol use the restriction enzyme Rsa1 primarily, and then BstU1 if that fails.. The recipe is simple –>

To this you add 20uL of 100ng/uL template DNA

We incubated this mix for 1 hour at 37C. We were hoping for a nice even smear that was mostly between 300bp and 1000bp. This is what we got…
So there are a couple of worrisome bands on the 1st 2 lanes labeled Degu, so we tried the BstU1 on that one. The others look OK, with the majority of the smear being between 300bp and 1000bp.

Here is the BstU1 digest.. Same master mix and incubation conditions.

So this is clearly much worse.. Most of the digest is well above 1000bp.. We decided against using this one…

At the end of the day Tuesday, we decided to go with all the Rsa1 digests, accepting that the band might cause us problems.. There is a PCR check on the next step that will allow up to determine the exact effect… Stay tuned…