PCR optimization for a long template

[care bear]

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ok, i know this forum isn't really for this purpose but i figure i'll give it a shot and if no one responds, that's fine too.

i'm having trouble amplifying a ~9kb fragment.
i'm using DyNAzyme EXT system for polymerase and buffer.

anyone have used this system before, or is a better one out there for long templates?

i have never tried to amplify a segment this long before. . .any thoughts would be helpful.

thanks!

 

[coldchemist]

I don't do a whole lot of molecular biology, but if I were in your position I would probably cruise around some websites (like Qiagen or Pierce) and look at other systems. I usually have a lot of success when I search the web pages of manufacturers.

 

[Treg]

There is a russian fellow in my lab who is an expert on taq and PCR. Here are his suggestions-

Use a mix of regular taq and pfu. Sometimes you have to try a few ratios to get it to work right.

Use PFU buffer.

Your primers should be a bit longer than normal, i.e. 25-30 bp (watch your G/C content).

the cycle you use should account for the longer time it will require during the synthesis phase as the reaction continues. This can be accounted for using a gradient machine.

You may have to toy around with your Mg2+ concentration.


This is all assuming that you are amplifying something from a plasmid. If it is from genomic DNA, it will be more difficult, but just expand on the possibilities of the above recommendations.

Good luck!

Treg

 

[tofurious]

Long PCRs are always a problem. We had some success with Takara Taq DNA polymerase from PanVera at Madison, WI. Like the previous poster suggested, you are highly recommended to use long primers and increased extension time. Note however that the longer the primer, the higher the annealing temperature is likely to be. Good luck!

 

[looseygoosey]

I would recommend Herculase (an optimized mixture of taq and pfu turbo). I was able to amplify an 11kb fragment with this polymerase. I believe that Stratagene manufactures it however it is very expensive.

 

[Aptamer]

Originally posted by Treg
There is a russian fellow in my lab who is an expert on taq and PCR. Here are his suggestions-

Use a mix of regular taq and pfu. Sometimes you have to try a few ratios to get it to work right.

Use PFU buffer.

Your primers should be a bit longer than normal, i.e. 25-30 bp (watch your G/C content).

the cycle you use should account for the longer time it will require during the synthesis phase as the reaction continues. This can be accounted for using a gradient machine.

You may have to toy around with your Mg2+ concentration.


This is all assuming that you are amplifying something from a plasmid. If it is from genomic DNA, it will be more difficult, but just expand on the possibilities of the above recommendations.

Good luck!

Treg

Like Treg said, play with your mag concentration. Give Invitrogen a try. They have a PCR optimization kit. It's actually pretty easy to use.

Where is this DNA from?

 

[care bear]

it is human genomic dna.

now i'm getting smears in most lanes. . .i've been playing around with the Mg ,the dNTPS and the amount of polymerase.

i have heard of making your own enzyme blend but it just seems like one more variable that i'd rather let someone else have control over; that why i was looking for opinions on the different 'long pcr' enzyme systems that are out there.

oh yeah, and i did switch to a 2 step program a few days ago that lets me increase extension time with each cycle after a certain number. . .

oh well, i'm hopeful and will stay at it 😉 thanks for all the advice.

 

[cmz]

http://micro.nwfsc.noaa.gov/forums/ has a good bit of information...

 

[Gut Shot]

I also recommend Herculase by Stratagene. It is indeed expensive, but whatever gets the job done the fastest is often worth it in the end. The quality of your template DNA may be the single largest source of your problems. I usually didn't bother with positive controls for normal PCR reactions, but you may want to seriously consider finding one if you haven't already.

Good luck!

 

[cmz]

Originally posted by care bear
ok, i know this forum isn't really for this purpose but i figure i'll give it a shot and if no one responds, that's fine too.

i'm having trouble amplifying a ~9kb fragment.
i'm using DyNAzyme EXT system for polymerase and buffer.

anyone have used this system before, or is a better one out there for long templates?

i have never tried to amplify a segment this long before. . .any thoughts would be helpful.

thanks!

Just to be sure about a few things, what are your cycling parameters for your PCR? What are your primer's Tms/Lengths/GC%? I would suggest that you just use PfuUltra (Stratagene/Biocrest) as it's a relatively cheap polymerase and works up to 19kb. Apparently, it might be helpful to titrate the amt of MgCl2 you use in your cocktail. Whatever Treg mentioned in his previous post is probably the way to go (basically the same thing I've echoed).

Also, I'm not sure if this will make a difference, but use 2 mins/kb of plasmid for your extention time.

 

[Aptamer]

Here's a PNAS paper from 1994...it's uses a combination of Tth polymerase and Vent polymerase (yes there is other thermostable polymerases besides taq and engineered taq. I'm sure you already have it done but it's there anyways.

Actually, the pdf is too big to attach so here's the link ->
PNAS Paper on Effective Amplication on Genomic DNA

Luckily, PNAS believes in an open access policy to science. Woo hoo!

 

[premed]

A 9:1 mixture of Taq😛fu is what I use. This also allows proofreading if you plan to proceed with cloning. You can PM me for any questions...

Mike