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We tried to purify some PCR-products in preparation for library construction for Illumina sequencing. For several samples, after mixing the beads and PCR-product, we ended up with what seemed like a low-percentage gel! The beads could not move to the magnet! The PCR products were amplified with the "gel stab" method, that is, two rounds of PCR, with the product from first run on a gel, "stabbed" with a pipette tip and used as template for the second PCR round. This was necessary because the first PCR did not produce a sufficient amount of product.
Another locus from a normal PCR was purified simultaneously and had no problems. So, we are thinking the small amount of agarose left caused the problem. We tried gentle heat to liquify the gel, with little success. Next we are thinking to add water and more beads (maintaining the ratio) in order to dilute the agarose. Has anyone faced a similar problem? Any ideas how to solve this?
Thanks,
Jon
Another locus from a normal PCR was purified simultaneously and had no problems. So, we are thinking the small amount of agarose left caused the problem. We tried gentle heat to liquify the gel, with little success. Next we are thinking to add water and more beads (maintaining the ratio) in order to dilute the agarose. Has anyone faced a similar problem? Any ideas how to solve this?
Thanks,
Jon