PCR Help - 2kbp product - (Feb/19/2008 )
See attached gels.
The top row of each gel is 3 sets of primers paired together in 9 ways (F1-R1, F1-R2, F1-R3, F2-R1,...)
The first one I ran:
1)94-3:00, 2)94-0:30, 3)58-1:00, 4)72-1:00, 5)Goto 2-40x, 6)72-7:00, 4-Hold
The second I ran:
1)94-5:00, 2)94-1:00, 3)60-1:00, 4)65-3:00, 5)Goto 2-35x, 6)65-7:00, 4-Hold
I increased the annealing temperature a bit, decreased the extension temp and lengthened the extension time (also fewer cycles, figured I was just wasting time).
I've seen some people say to use DMSO (up to 5%) in the PCR, but my area isn't really GC rich.... actually maybe it is now that I think of it, I might have to try that after all. Any other suggestions?
Sorry, what is the issue here? I can see that the top comb for each gel is a horrible mess of mispriming. The lower comb makes specific product for all but one lane in the first gel, and two smaller products on the second gel.
Do you want to get specific binding in the upper combs? What product do you want to generate? What targets do you have in the upper and lower combs, and in the two gels?
If you want a specific band, have you tried touchdown PCR? It will work well with repeating sequences, as long as your primers have been chosen properly. Make sure the 3' end is unique - if it has no homology anywhere else on the template, you'll not get mispriming events, no matter what the rest of the template looks like.
You talk about the GC content being high: what is yours?
So, I tried DMSO at 4%, didn't seem to change much.
The top rows of those gels is what I am looking at. I should be getting single products between 1500-2500bp. Only one lane really had anything over 1000bp. Don't want all the horrible mispriming. I think I may try your touchdown PCR suggestion.
Not sure what my GC content would be in the desired PCR products. I don't think it would be too terribly high. If it is, what kind of things can I try?
reduce the number of cycles to 30.
reduce the annealing time to 30 seconds. 60 sec is far too long. 30 second is generic start lenght.. also too long and can be trimmed down to as short as 10 secs.
What kind of polymerase are you using? I hope it is not taq. Taq doesn't amplify anything well pass 1.5kb. getting it over 1kb is difficult.
If the GC content is very low, consider dropping the extention temperature to 68 Celsius. It prevents the polymerase from falling off. (try other things first before considering this modification)
I assume you are amplyfing using genomic DNA as a template. Have you looked to see if your primers have multiple binding sites?
I BLASTed all my sequences, they should be good (however I want to sequence the area in between because we are suspect of its sequence, this is why I picked 3 different forward and reverse). I was using GoTaq Green so I am thinking this is probably not going to work. Currently running a touchdown PCR with it. I guess I will have to order some different polymerase. I ultimately want to sequence the product, so I guess I want some high fidelity polymerase?
What polymerase do people suggest? Promega T4? Finzymes? What do you think?
Also, what about raising the denature temp to 98? I saw some talk about that, what do you think?
Finzyme is kind of expensive... and too good to be used for this application. I am unfamiliar with Promega T4. I would use KODhifi... as I have used this enzyme before.
Denaturing temperature is somewhat dependent on the polymerase. Some polymerase can stand higher temperature then others. The higher the denaturing temperature the shorter in duration it is. Mainly an advantage for long PCR product.
I am not sure how taq reacts to 98 Celsius. Probably Taq won't appreciate it. But if you do try, drop the denaturing time to about 7 sec.
Finzyme is 'too good'? What do people use it for
Thanks for the advice!
"too good" as in "too expensive for routine PCR". It's used for applications that require higher fidelity than taq and mutagenesis reactions.
Probably DMSO won't help you if your GC is not too high. Consider the advice of Pernesseblue (taq isn't up to this length): most vendors have polymerase blends/polymerases that are suitable for more than 1 kb. If fidelity isn't an issue, consider the pricing! (in our lab people have worked with many different polymerases or blends over the years and most of them give amplification of products well over 1 kb).
I want to sequence the products, so I do want fidelity...right? I'll try KODhifi.
have you looked at the primers themselves... not where they bind.
Are they GC rich?
What are their temps?
Do they form stem loops etc?
Perhaps you just need new primers that are better suited.
If it is the polymerase, i'd also like to suggest Triplemaster.
Triplemaster is fantastic (just PCR'd up 7.4 kb with that).