Dear collegues,
The final aim is common: to band cut distinct PCR products from my gel slice after PAG vertical electrophoresis folowed by purification/elution. However, I could not obtain specific well defined bands in my gel in spite of well known capability of this approach to divide PCR amplicons few bp distinguished. I attached the picture of my gel (see the 3rd well). What am I wrong pursuing the ideal picture with defined sharp bands, whether it is feasible at all?
Gel composition: stock solution (20% acrylamide, 5% bis-acrylamide) diluted in 3.3 times, so the final concentration is 6%. Both Persulfate ammonium and TMED are used for crosslinkage.
Device parameters: Bio-rad chamber and power supply on impulsed protocol: 50V for 30' followed by 150V for 60'.
Product size: 870 bp
Some more information would help a lot to work out what went wrong here:
1) What's in the other wells? Wells 1-4 all appear to have DNA in them.
2) What buffer did you use to pour the gel and at what concentration?
3) What running buffer did you use and at what concentration?
4) What loading dye did you use?
5) Did you do anything to control the temperature during the run (eg cold buffers or running in a cold room)
6) What did you stain the gel with and how long for?
Can I also ask why you are doing this with polyacrylamide rather than agarose? Agarose is trivially easy (getting bands, and cutting/cleaning up your product) so I would only bother with polyacrylamide if I needed to separate PCR products that were really close together. I'm guessing there are other band/s in this prep that you don't want - how big are they?
Sorry for so many questions, DNA gels are just easier to troubleshoot with more details :)
Dear Laura,
I apologize for such late response but I ll do my best to provide you answers on aforementioned as clear as I can:
1) first well - ladder (inapropriate to use with PAG though it works), second and fourth wells contain the excess of loaded PCR product in 3rd well. I prefer to pour as much pcr content as possible to increase concentration of product on elution stage. The latter causes that wells might be overpoured on occasion because they are non capacious for such volumes (tupically 50 ul). In this case, only 3rd is overpoured
2) Do you mean type of dilution buffer used on initial gel preparation step? If so, 1x TBE buffer was used. I can find out distinct conc of any reagent If it needs. But, our technichans regularly make 5x TBE buffer as stock to be then used as someone wishes
3) Previous one was used as running.
4) The PCR was already colored by green dye (Promega GoTaq, Flexibuffer) prior to loading
5) No, I did nothing to reduce temperature. The entire run takes only 1,5 hours and whose 30' occurs just on 50V voltage, so the resulting temperature og the bath is not high.
6) I ussually stain my PAG gels by means of plunging into home-made "carrot" solution which is made on H20 basis with ethidium bromide supplement (approximately 5 drops of stock per liter)
As for least question, I think you are right and it might be the right option to switch on agarose. However, initially this technique was elaborated by others and I got it as the rule of thumb upon employment. I suppose those people pursued the aim to detect various mutations on the gene of interest including indels occur on occasion. So, to divide them simpler, particularly those distinguishing on several bp PAG was employed. It greatly increases ease on subsequent Sanger sequencing analusis. It is just my thought and I think I am very close to follow your advise. Nevertheless, prior that I'd like to clear this up.
Thank you in advance Laura
Hi!!!
As per the gel image, I found that there is problem with annealing temperature as the DNA quality is fine and highly concentrated in 3rd and 4th Wells.
To make it more flexible, it would like to recommend following things for the improvement:
- You should proceed with gradient PCR. Therefore, you can get the optimum temperature for the amplification.
- I don't know which marker you are using for this study... But if you are using SSRs marker then you can procced with 3.5-4.0% of metapho agarose.
I hope this will fix the problems in your experiment.
All the best!!!
Hello Anshuman,
thank you for your advice. However, may you clarify how does the primer's annealing temperature relate with existance of smear? My premise is if it related I would get several various length bands, wouldn't it? I completely fail to comprehend how inapropriate anealing temperature provides smeared DNA apart from both decreased efficiency and unspecificity.
Thank you loads!
Hello Alaxendar,
Yes, you are right. However, if annealing temperature is not optimized for the gene amplification then it indicates two scenarios:
1. There could be a high chance of non-specific amplication (as you already indicated this concern).
2. If primer binds to the desired gene sequence, there could also be a chance of amplication but due to underisred annealing temperature (but it should remain close to the desired annealing temp). It may get bind to the specific region with less affinity. This may lead to very light and absence of band (no amplification).
3. If I talk about the gel picture then only dense smear appeared. As we already know, primer consist stretch of nucleotides. So, improper amplification indicate the appearance of smear on gel.
Hope, this information is enough to clarify your concern.
Thanks.
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