double digest - trying to isolate a 450bp sequence with no sucess (Nov/28/2009 )

Hi, i am trying to digest a plasmid with two different restriction enzymes. BglII and BseR1 to be more precise. In general, for the first digest I use 1ug of DNA in a total of 20ul solution. I digest overnight and I have positive results. The problem is the second digest. I am unable to successfully complete it. The problem here is that the two enzymes require different digest environments. One is from Promega and the other from Biolabs. They have different buffers, so i can't make a double digest directly. I need to get that 450bp sequence after the second digest but I have been trying for 3 months without any success. Anyone can give me some tips on how to procede?

The problem is that after purifying from the gel, i always have very little DNA concentrations and i don't get the band i need on my 1% agarose gel.

Thx a lot

Sam

samal90 on Nov 28 2009, 05:33 PM said:

NEB suggests that you can perform a double digest in their buffer 3. Even though you are using one Promega enzyme, this should work fine. Use ca 5- 10 U enzyme/ug DNA for 1 hour.

If you don´t get your fragment after the digest it would indicate that one of the enzymes is not functioning. Can you linearize the plasmid when you perform a single digest with the individual enzymes?

If the digest does not work try to amplify the fragment you need by PCR and use it for cloning ...maybe the restriction site is mutated and one of your enzymes won't work anymore (this often happens when the fragment was inserted in the vector by the same restriction sites with whom you want cut it out!).

Regards,
p

samal90 on Nov 28 2009, 07:33 PM said:

Hi
You may digest the plasmid with the first enzyme, perform ethanol precipitation with glycogen carrier to minimize the losses, and then do the second digest with the buffer optimal for the second enzyme.
Furhtermore, I agree with klinmed, You should perform the digest with second enzyme alone to make sure it is functioning.
Good Luck
Michael

thx guys. I know that both enzymes are working properly. I've run some controls with both and they both linearize my plasmid on their own. I'll try to run the double digest in NEB buffer 3. I can't use PCR since my supervisor doesn't want me too or else i would've been done a long time ago lol

So, i guess i'll do the first digest in buffer 3 and for the second digest BseR1 requires Buffer 4. Do i add some to the mixture? or i just keep on adding buffer 3?

Do a single digestion, with both enzymes, in buffer 3. I would increase the total volume to 50 ul and keep the amount of DNA and the amount of restriction enzymes constant (but adjust the buffer amount, of course).

samal90 on Nov 28 2009, 12:33 PM said:

Since you believe DNA recovery is the problem I may have a suggestion for you. I have had very little luck with Qiagen gel purification kits in the past. What works for me is Ultrafree-DA columns from Millipore. Digest with enzyme #1. Excise your band of interest and place in Ultrafree column, spin 5000g for 10 minutes. Bring volume to 200ul with sterile water and EtOH precipitate DNA. Repeat with 2nd enzyme.

For peace of mind, you can quickly place your Ultrafree column on a UV box to see your DNA in flow-thru after spinning 5000g.

Good Luck