Hi everyone,

I am trying to purify a 200 bp DNA band PCR-amplified from the genome. The initial volume is about 2.5 ml (a pool of 50 PCR reactions). The plan is to first concentrate DNA into 150 ul elution buffer using Zymo Clean & Concentrator-100 and then purify the 200 bp DNA fragment by agarose gel electrophoresis. After the first step, I expected to see a significant enrichment of DNA (both the genomic DNA and the PCR band) as the 2.5 ml PCR product was concentrated into 150 ul. As shown in the attached figure (Lane 1: 1ul PCR reaction mix (from 2.5 ml) before clean-up; Lane 2: 1ul control PCR reaction mix (without genomic DNA as a template); Lane 3: 1ul concentrated DNA (from 150 ul)), I observed the enrichment of genomic DNA as expected. However, the 200 bp target DNA band was not enriched and actually, it was largely lost. I am very confused about this. The DNA Clean & Concentrator™-100 was designed to purify 50 bp~10kb DNA with a recovery efficiency of about 70~90%. I added 5 x binding buffer to the PCR mix as indicated in the manual. This binding buffer/sample ratio (5:1) is recommended to purify small PCR products less than 2kb.

The only steps that I didn't strictly follow the manual are that I centrifuged the column for 2 min (30s in the manual) and dried the column for 10 min (not indicated in the manual) to further remove the residual EtOH before adding 150 ul elution buffer. Another potential issue may be that the DNA loaded onto the column exceeded its max binding capacity (100 ug). There was about 50 ug genomic DNA in the 2.5 ml PCR product plus some amplified DNA fragments and dNTP. I am not sure whether this will cause an overload of DNA. Based on nanodrop result, the final DNA I recovered in the 150 ul elute is about 50 ug.

Any ideas?

Thanks in advance!