Agarose Gel electrophoresis - (Dec/07/2010 )
hey guys! i'm facing a problem on interpreting my result after gel electrophoresis. my school is doing restriction mapping and the plasmid we used is 9332 bp. but for a particular restriction digestion. we did a double digestion and actually we should get 3 bands 1282 bp, 4789 bp and 3261 bp. BUt the result we obtain after gel electrophoresis is 2000 bp, 4000 bp, 8000bp and 10000bp as the estimated value plasmid size which should not be the case after comparing with the DNA marker. i am sure i compared correctly. is there any possible reasons that this happen? my guess is that when undergoing gel electrophoresis, the agarose gel is place slanted causing the reading to be a bit off. I'm not very sure about this. Hope to get a reply soon! thanks alot 
DalalaTa on Tue Dec 7 18:01:08 2010 said:
hey guys! i'm facing a problem on interpreting my result after gel electrophoresis. my school is doing restriction mapping and the plasmid we used is 9332 bp. but for a particular restriction digestion. we did a double digestion and actually we should get 3 bands 1282 bp, 4789 bp and 3261 bp. BUt the result we obtain after gel electrophoresis is 2000 bp, 4000 bp, 8000bp and 10000bp as the estimated value plasmid size which should not be the case after comparing with the DNA marker. i am sure i compared correctly. is there any possible reasons that this happen? my guess is that when undergoing gel electrophoresis, the agarose gel is place slanted causing the reading to be a bit off. I'm not very sure about this. Hope to get a reply soon! thanks alot
Hi, I'm not sure if my reply will help but I'll take a crack at it. I can't tell exactly what's going on without looking at the gel, but perhaps you have incomplete digestion of one or both enzymes? Also, I'd take another look at the sequence to make sure that you're cutting what you think you're cutting. My steps would be something like: 1) resubmit sequence to NEBCutter 2.0 (http://tools.neb.com/NEBcutter2/index.php), tell the cutter that the sequence is circular and hit submit, then under main options hit custom digest, enter in your two enzymes, and then also get a profile for each enzyme used individually; 2) rerun the digest this time including a single digest with both enzymes (to make sure that each enzyme is working) and the uncut plasmid for comparison. Those are the steps I would take. I hope I've helped a little.....
~Nic~
DalalaTa on Tue Dec 7 18:01:08 2010 said:
hey guys! i'm facing a problem on interpreting my result after gel electrophoresis. my school is doing restriction mapping and the plasmid we used is 9332 bp. but for a particular restriction digestion. we did a double digestion and actually we should get 3 bands 1282 bp, 4789 bp and 3261 bp. BUt the result we obtain after gel electrophoresis is 2000 bp, 4000 bp, 8000bp and 10000bp as the estimated value plasmid size which should not be the case after comparing with the DNA marker. i am sure i compared correctly. is there any possible reasons that this happen? my guess is that when undergoing gel electrophoresis, the agarose gel is place slanted causing the reading to be a bit off. I'm not very sure about this. Hope to get a reply soon! thanks alot
The only thing that come to my mind is that probably when you ran your digests you used the circular ladder and that is what is giving you higher bands.... You might be getting four bands, because of incomplete digestion by both the restriction enzymes you are using, but I think you could do the digest again, run it on a gel again, making sure your ladder is correct. Hope it turns out well....