4 replies to this topic

[Red-eyed Rabbit]

Hi all,

sometimes I have this problem, but not usually. Usually  get nice bands after PCR (normal or long PCR). I use genomic DNA, usually 60 ng to a 20-30 ng reaction, so overloading can be excluded. now out of ~20 reactions, almost 10 was stuck in the well, while the others were running. Beside these I had plasmid controls (10 ng/reaction), this time they never stucked in the well, so this confirms it's not overload, for it is much easier to overload plasmid than a genomic DNA, so if plasmid was NOT overloaded that gDNA most possibly neither.
This was an optimization reaction for a long PCR (almost 10 kb), with Phusion polimerase (everyone loves it because it works very nicely). Interestingly, when I increased Mg++, I get more bands in a wrong size (small bands, ~600-800 bp). Plasmids usually gave the right size, but they usually had a smear beside (but still there is a very sharp, very visible clear band in the pale smear), except only for the high-Mg++ (3mM!) reactions (standard is 1,5 mM for Phusion). Temperature increase or decrease had no significant difference, if any, lower even worked worse (more aspecifis, small bands, and less bands in the specific sizerange, though those small bands of course are lighter).
We had a previous experimet, where we got many right-sized positives, but they had a very low yield, so optimization procedure was carried out for gaining a higher yield (I need the gelpic very much for presentation, without photpshop-cheating ). Any ideas?
1. how can I increase my yield, if temperature lowering, Mg++ increasing, DMSO titration and a buffer change did not help? Even the previously working reaction was fuly negative this time (I only used two of my samples, and two positive controls).
2. how can I avoid DNA stucking in the well? Does any buffer work better than other We currently are using TAE, I'm thnking of tryng SB (can be run on higher voltage, and this is a loooooong run...) do you think it can help? Or any other buffer suggestions?
yeah, I think that's all... any ideas are welcome.
RER

[Ameya P]

Red-eyed Rabbit, on 17 September 2010 - 12:10 AM, said:

Hi all,

Interestingly, when I increased Mg++, I get more bands in a wrong size (small bands, ~600-800 bp). Plasmids usually gave the right size, but they usually had a smear beside (but still there is a very sharp, very visible clear band in the pale smear), except only for the high-Mg++ (3mM!) reactions (standard is 1,5 mM for Phusion).

Well, giving too much Mg+2 to the reaction will allow polymerase to work at non specific places. That is why you are getting bands in wrong places. I would not bother too much with the recommendation of Mg+2, but would work around the concentration levels which work the best for me.

[Ameya P]

Red-eyed Rabbit, on 17 September 2010 - 12:10 AM, said:

Temperature increase or decrease had no significant difference, if any, lower even worked worse (more aspecifis, small bands, and less bands in the specific sizerange, though those small bands of course are lighter).
We had a previous experimet, where we got many right-sized positives, but they had a very low yield, so optimization procedure was carried out for gaining a higher yield (I need the gelpic very much for presentation, without photpshop-cheating ). Any ideas?

Usually, increasing temperature would give you more specific bands. But since, there is no difference in the yield, I think the temperature is optimum for the reaction. Keeping temperature constant, you should change other parameters in the reaction, like increasing time of extension, depending on your expected fragment size, Mg+2 conc. Also, I am hoping that your primers are brand new and not something that has been lying the freezer for years together.

With regards to the DNA being stuck in the well, I have never heard of that before and hope to learn something from the discussion that will follow.

Good luck!!

[keary]

Hi RER,
Did you ever figure out why the DNA was sticking in the well? I am having the same problem at the moment but it is genomic phage DNA not a PCR product. I have tried everything and anything to get it to move and it wont budge. I would be grateful if you let me know if you ever resolved the problem.

Cheers
K

[Adrian K]

can any of you post here your gel picture? I suspect is due to too much template loaded into your PCR reaction...