DNA damage in gel - (May/26/2010 )

Hi,
I was wondering what would be the maximum nm of excitation for DNA which will not cause any damage. I am going to use guanosine as suggested but want to know with out it.

Also, what are the other methods, if any to separate the restriction digested DNA apart from gel extraction. I am always losing a lot of sample from gel extraction and would like to know if there is any other method where I do not lose any sample.

Thanks,
Sanu

There are several kits especially for cleaning up DNA from a stained agairose gel.
For me the fastest way to get my band would be to use a tip of this kind:
http://www.cleaverscientific.com/products.php?viewid=41

You turn on the UV and quickly place the tip on the band. It doesn't take more than a second or 2.

I also found this site that may help you.
http://bitesizebio.com/2008/04/08/5-more-t...gel-extraction/

Good luck.

Maddie on May 26 2010, 03:23 PM said:

Thanks for your input. But are there any other methods which I can follow to bypass the extraction step?

If you have multiple products I don't see how you can bypass the gel extraction step. Is the product you want abundant? If it is you might be able to use a crystal violet gel which doesn't require UV, you can just look at the gel using a light box and take your time chopping out the band you want. The only problem is that it's not very sensitive and if your desired band isn't very strong you won't see it on the gel.

moerae on May 26 2010, 07:45 PM said:

I do have multiple products and having difficulty extracting them. I have been using the gel extracted DNA for ligation, but it did not work. So I was wondering if there is any other way I can do it. I do not think the crystal violet is going to work for me. Thanks anyway.

Hi Sanu,

How many products are you seeing on your gel? This is a stupid and time consuming way of doing it, but if worse comes to worse you can always just phenol:chloroform clean up your restriction digested DNA and use a bit of that for ligation. Transform and well, it's a matter of picking as many clones as possible and lots of colony PCR until you hit the one with the correct size insert. I know it's kinda stupid, but if gel extracted products doesn't ligate then this could be a last resort.

Out of curiosity, what kit do you use to gel extract?

moerae on May 26 2010, 11:58 PM said:

Hi,
After digestion, I get three products. I need two out of them to ligate. I am using the Genelute gel extraction kit from Sigma ( Cat No: NA1111-1KT). Here is the description of the products I get. I have a 9500 kb plasmid which is cut with EcoRI and DraIII to get a 9035 bp and a 466 bp product. On the gel, I only see the 9035 bp product and not the 466 bp product which is understandable because it has lower mass than that of 9035 bp.
For the other plasmid which is 4725 bp, i cut with ecori and draiii to get a 1235 bp product and a 3500 bp fragment. On the gel I can see both of them. From the gel, I am extracting the 9035 bp product and 1235 bp product and want to ligate them together but with no success. I did all the things that were suggested. I am going to do the ligation today and see if it works. Will keep you posted.

Thanks, Sanu

sanu on May 27 2010, 06:08 PM said:

Hi
It seems suspicious to me that You cannot observe 466bp product even on low agarose gel.
Did You make sure your double digestion is working properly?

Michaelro on May 29 2010, 02:48 PM said:

Not sure. Since the uncut plasmid is 9500bp and the cut one is 9000 bp,i thought there might not be much difference in the size. So I took the bigger fragment and went ahead with the ligation. Seems like my ligation worked because there are a few colonies on the plates.