Possible for standards to run differently from samples? - (Jun/12/2008 )
I've digested my DNA samples with 3 different RE's - DraI, StuI and PvuII.
The DraI digest pattern looks fine - get the expected pattern -but the StuI and PvuII is different. I see the correct number of bands but they are running next to incorrect weight markers - as if the standards are running slower than the samples.
I am using same buffer to load samples as with standards. Is it possible that samples could run slower or faster than standards?
My DNA samples were the product of a 3 part ligation reaction using unique cohesive ends - AflII, KpnI and XhoI.
The DraI digest pattern looks fine - get the expected pattern -but the StuI and PvuII is different. I see the correct number of bands but they are running next to incorrect weight markers - as if the standards are running slower than the samples.
I am using same buffer to load samples as with standards. Is it possible that samples could run slower or faster than standards?
My DNA samples were the product of a 3 part ligation reaction using unique cohesive ends - AflII, KpnI and XhoI.
I've had this happen before too. Here are some suggestions for you:
1) Check and double check your sequence map to ensure that it is correct.
2) Star activity can result from having too much glycerol (from the enzymes) in your RE digestion mixture. Ensure that you add no more than 5% RE to any digestion mix.
Thanks - I've double checked my sequence map and that is correct.
I'm adding 10% RE so perhaps it is star activity - when this happened to you did it turn out that you did have the correct sequence after all?
I'm thinking of just sending it off for sequencing so I can be 100% sure
I'm adding 10% RE so perhaps it is star activity - when this happened to you did it turn out that you did have the correct sequence after all?
I'm thinking of just sending it off for sequencing so I can be 100% sure
My sequence was fine. I just used too much BamHI which is extremely susceptible to star activity. Also,if you are doing overnight digestions, I would recommend you reduce the time of digestion to less than 4 hours especially if the DNA you are digesting is linear.
In any case, here is a link to NEB which explains star activity http://www.neb.com/nebecomm/tech_reference...ar_activity.asp.
I hope this helps.
The DraI digest pattern looks fine - get the expected pattern -but the StuI and PvuII is different. I see the correct number of bands but they are running next to incorrect weight markers - as if the standards are running slower than the samples.
I am using same buffer to load samples as with standards. Is it possible that samples could run slower or faster than standards?
My DNA samples were the product of a 3 part ligation reaction using unique cohesive ends - AflII, KpnI and XhoI.
The amount of DNA will affect where it runs. For your standard to accurately reflect size, it has to be at a comparable DNA concentration as your sample. Try running a couple different dilutions of either your sample or of the ladder.
This is common observation e-star and you have absolutely nothing to worry about. Your DNA is fine. Without knowing the exact reason why it occurs, i think the reactants are still bound to the DNA and that makes it heavier and thus causes it to migrate slower. It makes sense really. With everything we do to DNA, the enzymes in particular must interact with the DNA to modify it at some point, so they're going to be bound to the DNA. And if you run out a reaction without purifying the DNA (which is totally acceptable and purification should not be done), then there are still going to be reactants interacting or at least available to bind with the DNA. The marker runs quicker because it has been purified, it's just DNA. That's how i see the situation any way.