Hi,
I'm trying to find a way to remove as many as possible DNA genomic template from LR-PCR to ensure that subsequent nested-PCR are generated only from the LR-PCR template.
Gel purification won't fit, as we have many patients to run at once and, if possible i'm looking for a way to do it in batches. I thought it could be sufficient to dilute LR-PCR product, but does someone have another technique to propose ?
Many thanks for this answer. I will try to find the limit of dilution that can grant a correct amount of nested PCR without any contamination of genomic DNA.
The fact is that we use LR-PCR to isolate a region of gene which have a pseudogene similarity. Using very specific primers, i thought to be capable of increasing the gene sequence and dilution should have been sufficient to make rid of any traces of pseudogene. Unfortunately NGS sequencing gave me a different answer...
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