6 replies to this topic

[GoldEpi]

Hello,

I have tried to solve this problem but now my supervisor can no longer help!

I have the primers and the program from the publication and they should be ok.The only change is that we added restriction sites to 5' ends of the primers so they are 36 and 39 nt in length, and we use Phusion Hot Start polymerase instead of Hot star taq.

I have tried two different bisulfite conversion programs and still don't get any result. We thought something would be wrong with the primers so I measured them with Nanodrop and the other showed an abnormal curve (only one side of a hill with two slopes) and 260/280 of 2,2 and 260/230 of 0,8. A newly synthesised primer shows the same curve and values. Is it possible that inpurity interferes with PCR reaction? Is it possible that this polymerase can't achieve to make about 600 bp of product?

What would you do in my shoes? I'm happy to hear some suggestions and advice!

[Curtis]

I hope you realize that the Tm with Finnzyme's Phusion is different from Taq's. To calculate suitable annealing temperature use this online calculator:

https://www.finnzyme...ermination.html

[pcrman]

May I know why you add restriction site to the end of primers? cloning? why not TA cloning and sequencing? Adding restriction site to the primers makes already hard PCR much harder.

[GoldEpi]

Thanks for both of you!

To Metaller Scientist: That was a good point, I checked it out now. I have used 52 from the original program, and the two other methods give about 49. Now the primers (without restriction sites) give 62,45 and 56,98 in the red box. Such different values with the best parameters to Finnzyme polymerases! They say one should choose 3 C higher in my case. If I  choose 3 C higher from the higher one does the other anneal at all, I wonder... And Hot Start II should work in lower temperatures too.

To Epigeneticist: Thanks for the note! I have thought that too. My supervisor wants to use a high-fidelity polymerase and he doesn't believe that the restriction sites can affect the PCR. Do you or your collegues have some experience about this? Do you think ZymoTaq would be as reliable as Phusion Hot Start?

[pcrman]

First, you need to use some kind of hotstart taq polymerase, there is no need for high fidelity taq. Second, bisulfite PCR primers are already longer than regular primers, adding restriction site is absolutely unnecessary. Some people do add some extra bases to primers to give the pair similar melting temperature.

[GoldEpi]

IT WORKED with MyTaq! With Takara Taq I couldn't get a result but at least now I have one functioning cocktail and program! I really appreciate your help! Let us all have a nice weekend!

[Curtis]

GoldEpi, on 20 September 2012 - 11:16 PM, said:

Thanks for both of you!

They say one should choose 3 C higher in my case. If I  choose 3 C higher from the higher one does the other anneal at all, I wonder...

lt's alright, just keep it at the same temp, it'll work. I've tried before.