We isolated DNA from FFPE by manuel method. We done PCR but in gel there are some smear and we dont know how can we solve this problem. Can anyone have a solution?And why saw smear in PCR?
Hello, Ozge Uysal,
could you attach photo of your agarose gel? Smear could be degraded DNA (DNAse contamination) or nonspecific PCR products or smearing due to electrophoretic conditions (high voltage, bad buffer), etc.
It is hard to say without photo of gel
Hi Ozge,
I totally agree with Martin. Please attach a gel pic to get closer to your problem. But because DNA in FFPE tissues can be in really bad condition in terms of quality. Which depends on the age of the sample and the DNase inhibitor absence in the sample.
I think you have ran your DNA samples before PCR, haven't you? If the smear on the QC gel of the DNA. It related to degraded gDNA. Maybe you can send a brief protocol about your DNA isolation process, please.
Bests,
Tamas
This is our gel photo (the fifth well).
We use Xylol,etoh, TET (Tris, EDTA, Tween 20) . The protocol is Turkish if u want i can translate . Xylol to dissolve paraffin and we use TET to get DNA at the last step.
Hi Ozge,
This DNA seems to be totally degraded. If you have time and could you be so kind to translate it, would be good.
Thanks
Tamas
Hi Ozge,
I think you added too much template in your PCR system. So, you can try to reduce templete volume.
Good luck.
Hi Yanfen,
We had problem with PCR. We had to increase DNA concentrate during PCR. When we use less amount of dna , PCR didnt work. We have lots of problems :(
Hi Ozge,
I've checked the protocol what you had attached. To my mind it's okay for the deparaffinization and digestion process. But I more interested in your afterwards DNA isolation protocoll with your DNA quality check data (spectrophotometry (conc., DNA yield, A260/A280 and A260/A230 ratios) and the gelelectrophoresis picture of the isolated DNA before any PCR. Can you send it?
Do you use phenol-cholorform or high salt method for DNA isolation? I've got a really got protocol for DNA isolation by high salt method but for normal tissue samples only. It can be modified to fit after your FFPE protocol. You can reisolate the DNA from FFPE tissue.
Or if you have the DNA already, and don,t have more FFPE sample to isolate from them. But the DNA impure (A260/A230 ratio
Hi Ozge,
I have attached both protocols. After you have good DNA in terms of quality and quantity try PCR again. If it fails again then try this.
I suggest to do a touchdown PCR. If you can use PCR machine available with this feature. For the annealing temperature just set the highest temp for exmple 65-70 °C. Set the PCR to decrease the annealing temp by 0.5 °C after every cycle down to the calculated annealing temp of your primers. For example 55 °C (lowest temp). Run the PCR on this temp for the following calculated cycle number. For example: (total cycle number)-(numbers of decreasing temp cycles) = (remaining cycle number). E.g.: 40 cycles - 20 cycles = 20 cycles at 55 °C. This technique not as elegant as gradient PCR, but it can be usefull. Unf you won't know the ideal annealing temp like in grad PCR, but it will maximize the amount of the specific product.
Cheers,
Tamas
Thank you very much for your help. we will try these techniques and your suggest. ı hope we will do it. thanks a lot.
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