Hello,
I am working with eDNA for my masters project. Very briefly, the workflow includes an amplicon PCR which uses primers with illumina indexes, followed by a barcoding PCR, which includes the illumina i07 and i05 barcodes. After the amplicon PCR, we have a lot of primer dimer, which is interfering with the subsequent barcoding PCR. I am working in 10ul PCR reaction volumes, and would like to perform a PCR cleanup of some kind to remove the ~100 base pair primer-dimer, and excess primers. Because of the large number of samples (570) I would like to do this cleanup in a 96 well format. Does anyone have a recommendation for PCR cleanup kits that will remove ~100 bp dimers, are compatible with 10ul volumes, and are in a 96- well format? Our lab does not have the Qiagen vacuum which is required for 96 well MinElute cleanup kits.
Alternatively, our lab does have AMPure XP beads, but my initial attempt at using these to do the PCR product cleanup did not work well. After the Ethanol wash/dry step, the beads would not re-suspend in TLE, and when placed on the magnet stand, would not collect on the magnets. I am guessing that the beads were allowed to dry too long, but I am not sure how to go any faster in a 96 well plate. Anyone have any tips/tricks to avoid this? As a further complication, I am using BSA in the amplicon PCR, which some have mentioned will cause the beads to clump.
I am working with eDNA for my masters project. Very briefly, the workflow includes an amplicon PCR which uses primers with illumina indexes, followed by a barcoding PCR, which includes the illumina i07 and i05 barcodes. After the amplicon PCR, we have a lot of primer dimer, which is interfering with the subsequent barcoding PCR. I am working in 10ul PCR reaction volumes, and would like to perform a PCR cleanup of some kind to remove the ~100 base pair primer-dimer, and excess primers. Because of the large number of samples (570) I would like to do this cleanup in a 96 well format. Does anyone have a recommendation for PCR cleanup kits that will remove ~100 bp dimers, are compatible with 10ul volumes, and are in a 96- well format? Our lab does not have the Qiagen vacuum which is required for 96 well MinElute cleanup kits.
Alternatively, our lab does have AMPure XP beads, but my initial attempt at using these to do the PCR product cleanup did not work well. After the Ethanol wash/dry step, the beads would not re-suspend in TLE, and when placed on the magnet stand, would not collect on the magnets. I am guessing that the beads were allowed to dry too long, but I am not sure how to go any faster in a 96 well plate. Anyone have any tips/tricks to avoid this? As a further complication, I am using BSA in the amplicon PCR, which some have mentioned will cause the beads to clump.