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

Some of the groups in my institute are moving away from kits etc completely, and just crush the gel slice, make up to 1 .5 ml with TE and leave in the fridge overnight. The next morning they spin down the agarose, remove the S/N and do an ethanol precipitation. Slow, I know, but it's faster in the long run. If the DNA product is smallish, use ammonium acetate rather than sodium acetate: 1/3 vol 7.5M ammonium Ac, 2 volumes of the new volume EtOH.

Thought for the day.................are you sure that your PCR product doesnt have any internal BamHI sites? You may be purifying your product and (if you are going straight into your digestion step) may just be cleaving it to pieces!!

Can I do like that !!!because I have to ligate it before and for that minimum amount of DNA is required 100 ng.

mastermi on Jun 9 2009, 01:49 PM said:

It sound good but does it cause any problem in firther cloning assay
thanks

swanny on Jun 9 2009, 06:52 PM said:

xyz on Jun 11 2009, 03:56 AM said:

There shouldn't be any problems at all. Once you have done the EtOH precipitation, it's as clean as DNA prepared by kits. Just make sure you get rid of trace EtOH, by leaving the tube open and inverted for a few minutes. You can also heat gently for a few minutes to make sure.

Can you please provide me the exact protocol of it.
Thanks

swanny on Jun 10 2009, 06:37 PM said:

xyz on Jun 10 2009, 07:53 PM said:

100 ng is waht the protocol says, and that is probably the best way to make sure that ligation does work.
But that doesn't mean that it doesn't work below that.
I have often ligated fragments that I haven't seen on the gel, and most times it worked out well (maybe I had just 1 out of 24 instead of 12 out of 24 right clones).
Just take less amount of your vector to make sure your vector-fragment-ratio isn't too bad.

Thanks a lot. what do you suggest for the vector DNA ratio.

mastermi on Jun 11 2009, 06:30 AM said:

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?
Just one suggestion from my side.

Well,
I was trying to digest with MBO1, bands should be of following sizes
Undigested band: 152
Digested: 125 and 27

In agarose gel, i never saw the 125 band for unknown reason, so was thinking digestion did not work, 1 week i wasted.

Then i ran 12% Native PAge which is good for 50-200 bp DNA fragments.
saw two bands, one at 152 and other at 125 in digested sample and only 152 in undigested one.

May be it is mysterious, but worked for me.

xyz on Jun 11 2009, 07:02 PM said:

When I have so poor fragment, I ususally don't waste any for measuring its concentration but take all of it for ligation (elute it in the volume you can maximal put in your ligation). I then simply use 1/4 of the amount of vector I normally use. That would still be enough colonies after transformation...