ExoSAP-IT question - troubleshooting (Sep/01/2008 )
I browsed the forums for this product but did not find the answer I need. I have been recently trying ExoSAP-IT to clean up my PCR products in a 384 well plate. It works to a fair degree, but the products are not as clean as I hoped. I also compared ExoSAP-IT PCR cleanup to bead purification and the beads actually are working better than ExoSAP. My protocol is thus: I do PCR on my DNA, run a gel on a small amount to see how the amplification is. The amplification always works. Then I clean up with beads versus ExoSAP, run another gel to see how cleanup is. Here, beads work better than ExoSAP.
I want to troubleshoot the ExoSAP-IT vial I have to see if this is the problem. Someone in the lab suggested to prepare my PCR reaction as usual, but do not add in the Taq enzyme and don't do PCR. Just run a gel on a small amount and you should get no product. Then treat with ExoSAP and run another gel to see if ExoSAP is doing something else. I am not sure if this will actually determine if the vial I have is good or not. Anyone have any ideas?
We have used exosap-it a lot to purify PCR products before sequencing them and are generally satisfied with the results. By running PCR after ExoSap reaction, what do you expect to see?
After comparing the bead purification to ExoSAP treatment, we see the PCR bands are cleaner on the gels from bead treatment. We also sequenced these same bands and saw that DNA sequences from the bead treatment had lower background versus exosap, which is what we were aiming for in the sequencing data. So we are wondering why our exosap cleanup is not performing as well as the beads. So, I want to see if it is the exosap I have and what ways to test if this is the problem.