Here, I did long-range PCR for ranges between 8-24Kb. I used 0.4% agarose concentration but the PCR stuck to the wells and some of the parts migrated.
I am not sure if this is the same products in which it got separated or not.
I used 30 cycles. The DNA concentration for the first three lanes about 300 ng per 50 ul, while the second three lanes (the same samples) about 700 ng/ 50ul. I increased the DNA because as you see the first three lanes give me faint bands.
If I change the DNA comb to a bigger size, is this going to be help? Especially for the long-range PCR?
Thanks a lot in advance.
Dear Halawani
Plz accept this book as a gift from me. it will help you a lot for PCR and downstream procedures.
Best Regards
Hi,
The first lane is the marker. And all the the 6 lanes from the same DNA template but different targets. The first three lanes are the same targets as the second three lanes with different DNA concentrations.
looking from the markers, I dont know the range of your makers, at least those two fat bands look like primer dimers. while other two lanes, show some PCR product but its unclear, if you know the expected length of the PCR product, then its easier to judge. otherwise, I would send them for sequencing.
If you used too much of the DNA Template, that might be the stuff staying in the wells. I had similar issue while I was doing PCR from genomic DNA. I would always load a sample of my template to compare, because it might also be a very long PCR product and hard to distinguish from genomic DNA.
As said above, the changing comb won't help but loading less might.
What kind of DNA did you use as template and how much did you use as template for the PCR? The reason I asked is that it could be that the faint bands are not PCR products. But this would be easy to test with just using the template in the same amount on the gel as comparison.
And also what kind of ladder did you use? Even if it did not seperate nicely it seems to me that you need also an additional ladder for the "smaller" PCR product.
For long PCR products you would get the best electrophorese results with a pulse gel electrophoresis. We don't have one available therefor we use 0,6-0,8 % agarose gels and then let them run very, very slow with just 10 to 20 V for 24 h up to (really!) 48 h and stain them afterwards with EtBr. This slowly we can get a nice separation even from bands above 20kb up to nearly 40kb.
Dear Halawani
Plz accept this book as a gift from me. it will help you a lot for PCR and downstream procedures.
Best Regards
Dear All,
Thanks a lot for your reply. I run another PCR that gave me good PCR as well as good sequencing results. I switched to another polymerase which has higher fidelity.
Running different targets showed that it might the problem basically from the polymerase or from the different conditions.
I tried to load less DNA up to 5ul rather than 10ul but I am still get the DNA stuck to the well.
A special thank to Sibtain and definitely I've accepted your gift :)
The second experiment I did that I mentioned with 12 Kb products. I did get good PCR products with another polymerase. Here is the photo. I used another marker which was separated well. The products should be lower little bits against the marker !!
Anyway, this smaller target, lower loaded PCR (5ul) and did get the same results.
One thing I noticed in this protocol and that the issue that changed my mind to switch to this polymerase is that it reduces the time efficiently.
The PCR condition does not have any initial denaturation or final extension.
The condition as following:
30 Cycles of (98 C for 10 seconds and 68 C for 10 minutes).
It looks very strange but the PCR before work better but now I get the bands stuck to the well. The only thing that I believe it caused the problem is that there was insufficient denaturation for the DNA template so due to that it stuck to the well.
Thanks.
Amr
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