5 replies to this topic

[umam]

Hi,

I have a 69nt insert (PCR product). I used 5 prime gel extract kit and cleaned it up for liagtion with pGEM but after running a gel with the purified product, I don't see a band. I tried this 5-6 times, even using the Promega kit and using liquid PCR product instead of gel extraction, but it does not seem to work. Any ideas for cleaning up such a small product?

Thanks,
-Uma

[fishdoc]

umam, on Jul 28 2009, 04:24 PM, said:

Hi,

I have a 69nt insert (PCR product). I used 5 prime gel extract kit and cleaned it up for liagtion with pGEM but after running a gel with the purified product, I don't see a band. I tried this 5-6 times, even using the Promega kit and using liquid PCR product instead of gel extraction, but it does not seem to work. Any ideas for cleaning up such a small product?

Thanks,
-Uma

I don't know about the kit you're using, but I think the Qiagen kits I use have a cutoff of about 70 bp or something like that. Your product may be too small to purify that way.

Furthermore, purification kits are known to be pretty inefficient. Did you start with a PCR product that had a lot of amplicon? If so, I would call up your Promega rep and ask about the lower end of base pairs the column will purify.

[Rsm]

Hi,
I have used the Qiagen QIAEX2 kit for cleanup of very small (and very large) PCR products (and targeting vectors). So far it worked pretty well.
However, the recovery rate is bad. Alternatively, you can clone your insert directly into your vector (overlap extension PCR). I would recommend that.
Cheers,
Minna

[eldon]

69nt?

synthesize it.

[perneseblue]

eldon, on Jul 30 2009, 06:03 PM, said:

69nt?

synthesize it.

I agree.

Just buy two oligoes that are complementary to one another. It is faster that way. Buy them 5-phosphorylated if you want to avoid the hassle of self-ligating vectors.

YOu could also design the oligoes so that once they anneal to each other they form staggered ends.

[Penguin]

eldon, on Jul 31 2009, 03:03 AM, said:

69nt?

synthesize it.

MWG will synthesize this length of DNA for around £0.04 per nucleotide (operates only in Europe I think).  I have just done this to generate a 52bp insert which I have successfully ligated into my plasmid
P