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
Can your restriction enzyme bind to the DNA fragments after digestion? Check the information provided by the manufacturer. If that is the case, use DNA loading buffer containing SDS in order to disrupt the protein DNA-interaction so you make sure that bands that run between the markers 1kb and 4kb are actually fragments larger than 1kb.
Good luck
I expect that most of the short fragments are from the small end of the gel possibly different shaped molecules running as larger than their true size. Possibly also some bias during purification of the dna. Is the answer simply to take the 2kb-4 kb slice of the gel hopefully removing the shortmers selectively?
Can your restriction enzyme bind to the DNA fragments after digestion? Check the information provided by the manufacturer. If that is the case, use DNA loading buffer containing SDS in order to disrupt the protein DNA-interaction so you make sure that bands that run between the markers 1kb and 4kb are actually fragments larger than 1kb.
Good luck
You should change your conditions of gel electrophoresis since you run much to fast to have a good resolution. It should work perfectly well if you run at 25 V for 6-8 hr. You may even increase the resolution if you run overnight in the cold room.
Best
It is difficult to do a genomic library. The best way is to use the phage lambda that
is more efficiency that a plasmid. What is your goal ? May be you can have another approach more easy
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