pcr issue-gene not amping - (Jan/06/2008 )
Hi,
I am new to using molecular techniques and am having a pcr problem, any suggestions would be quite helpful. I am currently working with Cyt. b. in snakes. I have been using DNeasy kits to extract the DNA from tissue samples (typically muscle tissue that is kept at -80, but also shed skins). I have had pretty good luck so far, but have recently had an issue with a particular tissue sample that is important to my project. I have used the DNeasy spin columns 2x now to get a DNA extraction. The first time I ran the extraction out on a gel to confirm there was DNA in it. There was. My pcr worked. However, when I went to sequence it, nothing...so I tried it again...the pcr did not work. So I tried a third time...still nothing. I re-extracted it last week, ran out the extract, there is DNA in it...pcr still doesn't work. I have tried it using both whole gene primers and also using internal primers specific to the snake species. My question is, is it possible I didn't get the EtOH out of the extract and that is the issue? This extract is from a shed skin, which had a lot of dirt, etc. on it, so the extract isn't even totally clear, it has a slight tinge to it. Is it possible there is too much DNA in the extract an that is why it isn't working? What would the signs of this be? The pcr shows zip...I ran several other extracts at the same time which worked, so it's not anything that has gone bad reagent-wise.
Thank you!
Sara
I am new to using molecular techniques and am having a pcr problem, any suggestions would be quite helpful. I am currently working with Cyt. b. in snakes. I have been using DNeasy kits to extract the DNA from tissue samples (typically muscle tissue that is kept at -80, but also shed skins). I have had pretty good luck so far, but have recently had an issue with a particular tissue sample that is important to my project. I have used the DNeasy spin columns 2x now to get a DNA extraction. The first time I ran the extraction out on a gel to confirm there was DNA in it. There was. My pcr worked. However, when I went to sequence it, nothing...so I tried it again...the pcr did not work. So I tried a third time...still nothing. I re-extracted it last week, ran out the extract, there is DNA in it...pcr still doesn't work. I have tried it using both whole gene primers and also using internal primers specific to the snake species. My question is, is it possible I didn't get the EtOH out of the extract and that is the issue? This extract is from a shed skin, which had a lot of dirt, etc. on it, so the extract isn't even totally clear, it has a slight tinge to it. Is it possible there is too much DNA in the extract an that is why it isn't working? What would the signs of this be? The pcr shows zip...I ran several other extracts at the same time which worked, so it's not anything that has gone bad reagent-wise.
Thank you!
Sara
PCR is one of those expts where the saying "less is more" can be used. Try repeating the reaction with a 1/5th and a 1/10th dilution of template DNA (what volumes are you using, by the way?) and see if the result gets any better. This could help in one of two ways: if there's too much template the reaction should work better (but there area number of conflicting opinions on that point). Secondly, if you're concerned about contamination, a diluted sample might sufficiently dilute any contaminants that they cease to be a problem.
Failing that, I'd try a repeated ethanol precipitation of the sample.
Thanks, I'll give that a try and let you know how it works!
Swanny is spot on about too much template and diluting contaminants. However, given that you got nothing on your gel, that more suggests contaminants. Too much DNA usually gives a faint band of interest and a smear above and below the band and possibly other non-specific bands on the lane. Are you aware of GC-richness and the affect that has on PCR? If your gene is GC-rich (>55%) your PCR would probably benefit from adding some betaine (0.5 - 2.0 M). That may improve the efficiency of the PCR and more easily amplify your gene. I also remember something about humic acid in soil samples. I think it inhibits PCR and is relatively simple to remove, although you may just dilute the template to remove that. Don't quote me on that one. To answer your question, i guess EtOH is a possibility, although an unlikely one. If you're worried about that then just make sure you get rid of all of it. There's probably another reason why it stopped working, or rather you got lucky the first time.
Nice post and good luck,
Rob