Biology Forum › Molecular Biology › Restrictin digest HELP!!
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- October 15, 2005 at 2:08 pm #2165
BioTechParticipantHi all… a quick question for you. I set up a single restriction digest using EcoRI, BamHI, and HindIII on lambda DNA. The protocol that I followed is for a 30 min. incubation at 37C with a 10 microliter reaction volume. Well, I was in a hurry to leave work yesterday and I forgot the digests in the incubator… total incubation time was roughly 14 hours!! ๐ณ This experiment isn’t for research; it was only a pre-run for a classroom exercise. I used 4uL of DNA (0.1ug/uL), 5 uL of 2X buffer, and 1uL of each enzyme. So, my question is whether or not you think I should even bother running a gel on these samples or if I should set it up again. Any input is greatly appreciated!! :)[/list]
- October 15, 2005 at 2:16 pm #30967canalonParticipantquote BioTech:Hi all… a quick question for you. I set up a single restriction digest using EcoRI, BamHI, and HindIII on lambda DNA. The protocol that I followed is for a 30 min. incubation at 37C with a 10 microliter reaction volume. Well, I was in a hurry to leave work yesterday and I forgot the digests in the incubator… total incubation time was roughly 14 hours!! ๐ณ This experiment isn’t for research; it was only a pre-run for a classroom exercise. I used 4uL of DNA (0.1ug/uL), 5 uL of 2X buffer, and 1uL of each enzyme. So, my question is whether or not you think I should even bother running a gel on these samples or if I should set it up again. Any input is greatly appreciated!! ๐
If you do not have limited time or product availability, it could be better to redo the digestion (15 min max to set it up and 30 min to digest) less than 1h lost isn’t woth the time loss to run the gel, analyse result, be sorry because of star activity (non specific digestion) and redo the digest anyway. But you can always put this one on the same gel as the new digest just to see if it was worth it ๐
ANd you may have too much enzymes (3ul in 12ul if I read you correctly, meaning that your buffer concentration is not OK either) in your mix. I have always avoided having more than 10% of final volume of enzyme. The storage buffer can have weird effects on your reaction. Complete volume with water.
Patrick
- October 17, 2005 at 6:26 am #31033BioTechParticipant
Hi Patrick… thanks a lot for your response. I set up separate reactions for each enzyme, so only 1 enz. per reaction tube. My main concern was star activity, but I didn’t see any indication of this happening when I looked at the paperwork that came with the enzymes. But, anything’s possible, right?! Another concern of mine is that I don’t have only one reaction to set up for each enzyme; I’ve got some old enzymes that have no dates on them, so I was also testing for activity. I have 4 tubes of EcoRI, 6 tubes of HindIII, and 6 tubes of BamHI, and I’m also testing 3 samples of lambda DNA (two of the tubes weren’t stored properly by the old tech and one is a brand-new sample that I recently received). I probably don’t need to test the new DNA, but I’m somewhat of a thoroughness freak when it comes to testing things. Just to clarify, I had 4ul of 0.1ug/ul DNA, 5ul of 2X buffer, and 1ul of enzyme for at total of 10ul in my reaction mix. Any more input is welcome!! ๐
- October 17, 2005 at 1:52 pm #31057canalonParticipant
In Your case I would be concerne with 2 things:
– Star activity for the enzyme after O/N digestion. But more than quantity it could depend on concentration (How many units reprensent 1ul? It varies between enzymes, manufacturers etc..). But in my experience [/i]EcoRI and BamHI (I don’t remember using a lot of HindIII ) are quite safe. But In my experience again, enzymes that are kept at -20ยบC and handled properly can go much longer than their expiration date.– Is there enough of your old DNA to be detected. Concentration is what is read on the tube, or did you measure it? DNA is usually stable, if freezed and thawed often it start to degrade, so at least a control with the new tube would tell , in case of problem why the results are looking bad.
Hope this helps
Patrick
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