No product after restriction digestion - (Jun/08/2009 )

Hi all,
I amplified my PCR product and it gave a nice band (4 microgram conc around), after that I puified it with qiagen kit and digested it with BAMh1 enzyme , after that I didn't get any band on gel. What could be the reason. I tried both qiagen I and Qiaex II kit for purification but no yield. Need suggestions.

Purification didn't work?

did u run a gel to check if you have your products after purification and before the restriction digestion?

Just one suggestion from my side.

As after purification with Qiagen kit, the concentration lowers , so check the concentration, run the gel. If you see the band, Then take appropriate amount to digest.
After digestion, sometimes instead of running agarose gel, for small DNA fragments run Native Page. It helped me a lot.

Thanks,

Hi
Thanks for suggestion. What is the rationale for native Page( poly acrylamide GE). and one more question is that checking the conc with spec (Nanodrop) makes any sense?

epigenetics on Jun 9 2009, 06:10 AM said:

No I didnt do that. Just after purification digested the product

jiajia1987 on Jun 9 2009, 12:24 AM said:

xyz on Jun 10 2009, 12:53 AM said:

you should have done that because it might save much time for you.
ps: bamhi has star activity from time to time at a high concentration.

Thanks.
Star activity depend on units of enzyme to conc of DNA , glycerol concentration and some other factors as well.I am using only 1 microliter of enzyme and that gives only 20units/microgram of DNA, so I am assuming that star activity is not giving problem to mu experiment.
If is there anything that I am not getting about star activity

medivh on Jun 9 2009, 09:12 AM said:

Although you don't see anything on the gel: Just try to clone it.

And if that doesn't work: Avoid using gels and kits! Make phenol-chloroform-extraction or only ethanol-precipitation instead...

xyz on Jun 10 2009, 02:03 AM said:

Sometimes, it is good not to assume. I use BamH1 in my work and it has a lot of problems (even if I see a clear and nice band after purification and after digestion).

And, mastermi is right, try to cloen it even if you don't see anything on the gel. I experienced this before and simply extracted the DNA from where I think the band would be (you know the size of your insert) and cloned it and it worked.