For the past two months I have been trying to analyse my AFLP regions on a 6% UREA PAGE GEL. The sample are selectively amplified with 4 set of primers.I have tried to optimize the protocols for 6% gel and as well i have tried several LD (2, 5 and 6 X LD). After the PCR I take 5ul of sample and 5ul of dye, at 95C I heat the samples for 5 min to denature and place them on ice before loading. I usually load 5ul of sample-LD mixture. For the gel I do a prerun for 30-40 minutes in a 65C 1x TBE buffer. With the samples I run for approx. for 1-1.5h at 60V ( the gel casting tray is 10x10cm). I have tried as well other options, longer preruns, longer runs and I added as well in the selective PCR some BSA but still I keep getting same results. These are pepper samples (genomic DNA). I do also take care that the APS is fresh and other chemicals are purchased from Sigma aldrich so I disclose the possibility that some chemicals are not properly working. In PCR I amplify the positive control. Please help. Gel picture is attached, on gel are several pepper samples.
Hi, I have never seen photos like these of PAGE gels...I usually scan my gels in a normal scanner and see the bands with nice resolution. Where is the molecular weight marker there? What is the size of your cameras and glass for silver staining is 10 x10 also? What is the weight range of those bands your are showing? I see also that bands are not well separated... by LD you mean the loading buffer? I think that perhaps yes, your gels are very small, as many bands are expected, there are other cameras and glasses for this gels, that are quite big... like 50 x 50 cm or something like that i do not remember.
The fragments are of size (100-700bp), its EtBr staining of UREA PAGE gel.
The scanner is from BIO RAD ChemiDOx XRS+, I assume that one of the reasons may be the small gel size but still nice bands shall be visible. LD is loading buffer.
Hello, this is not my forte but I'm adding my bit. As Diana asks, where is the DNA ladder? If you run a gel with the ladder and it looks smeary, the problem is the gel, but if the ladder looks OK the problem would be in the PCR itself. For 100-700 bp I would run a 2.5% agarose gel, and see whether you still get those smears (in which case the problem would be in the PCR reaction).
Best,
Ruben
Hello, how many bands are you expecting of each combination? I remember when i worked in Rubus that I observed in average about 30-35 bands per each combination...you have to assess if really with this small gel you will be able to see and compare the size and the quantity of bands. One rationale of a bigger camera should be to obtain a nice separation of bands to have a precise scoring of the bands in the gel. Other point is the ethidium bromide staining, i think that because you have many bands it should be better and most common in this kind of analysis the silver staining because it provides a better quality of the gels to read. I would also suggest to find a bigger camara for running your samples...
20 -30 bands..I agree that the gel is small, but as far I know the smear shall not be there (even in a smaller gel).. Today i did 8% gel, ethidium bromide staining, with 2xLD and 5X LD and with different DNA concentration but i got identical band, with a lot of bands ? Could the setup in PCR be a reason for these smears? What if I run an 1% agarose gel?
Thank you Artur. Well, i have used several different protocols and mostly all of them are explained in a similar manner. I have attached two protocols (from JOVE and from Thermofisher). Why i do denaturing, because all of the protocols that explain AFLP for the pepper analysis do denaturing, maybe its not an valid argument but it was a sufficient reason to do the same. I use the BioRad mini protean tetra system for the gel. Now i am thinking to use silver staining and to make new buffers and reagents with 6 and 8% urea gel.
Hello, as I mentioned PAGE is not my forte, but I thought that the stacking was just for proteins, and you use the difference in acrylamide concentration and pH to stack the loaded sample against the running part of the gel, so as to maximise resolution of the separation. However, I thought you never use stacking for DNA gels... although it's slightly off-topic, would you mind giving me some insight on this, Arthur?
There is no positive quality control for the gels, and we are assuming that the gels are the problem, but without a ladder (or a good concentrated agarose gel) we still don't know whether the PCR product is the culprit...
Best,
Ruben
I have not used stacking for the DNA UREA PAGE GELS..Today we have done UREA PAGE again, using the Selective PCR samples with only one set of primers, so same sample three times and each time with a different primer. We loaded the samples with 5X LD and the results can be seen on the attached picture. Again we got a lot of smears....we preheat the 0.5M TBE buffer up to 60C and do prerus for 30 min as well. The gel is 10 x 10cm. Any suggestions? Could the reason be that the ligation didn't work well ? Or eventually preselective PCR with the adaptor sequences ?
I agree with you as well. I will follow your instruction and in the same time i will do the restriction and ligation procedures again with 3 samples, with preselective and selective amplification and finally load them on 1.6% agarose and 6% urea polyacryamide. Best regards
Could you finally solve it Jasmin. Because I get similar band pattern in my denaturing Urea-PAGE. Please let me know if it worked out in any of the ways.
Thank you.
Martin Pecasa gave the right suggestion, it worked with me as well.
- Badges
- Science topic
- Similar topics
- Biological Science
- Genetics
- Genetic Analysis