Hi everyone,

I perform a PCR + Restriction Digestion (single cut, same at both sides) over a metagenomic library. After that, I am trying to purifiy DNA fragments between 1-4kbp long from the smear I obtain after running the PCR in an agarose gel. Agarose gel is 0.8% and I run it at 100V 40 minutes. I carefully cut the band between 1-4kb and the yield or DNA recovery is quite good. The problem is that, after cloning it and taking a look at the size of the inserts, 90% of them are much smaller than 1kb.

What would be the best DNA Electrophoresis conditions (% agarose, Voltage, time ...) in order to maximize the separation between 1-4kb fragments from the smaller ones?

I believe that even a small quantity of small fragments after purification will be over represented after cloning protocol so, every tip in order to get rid of them as much as possible will be very welcome.

For tIn the ligation step I tried different ratios vector/insert (3:1 ,1:1 and 1:3) and temperatures (RT, 16 and 4ºC). The size distribution is basically the same.

Thanks a lo beforehand

Jorge

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