How to amplify large fragment by PCR - SOS (Nov/24/2004 )

I am trying to clone a 4kb DNA fragment from genomice DNA of chlamy. Chlamy DNA is GC rich, so I added DMSO but it didn't work. I also used BAC to be my template, but failed again. I have checked my primers(including hairpins or false priming site, and I changed different cycle numbers, different annealing temperatures, expanding temperatures.

If you have experiences on amplification of large DNA fragment, would you like to share with me? I appreciate your help.

I'm amplifying a fragment which is about 8 kb long. I changed to the system from Qiagen. It's the protocol for amplifying large fragments with a combiantion of Proofstart Polymerase (Proof reading enzyme like Pfu but from Qiagen) and Taq Polymerase. Befor I really had difficulties to get a clear fragment without other bands. Now I get a defined band with good quality and pretty much dna. I hope I could help you.

Thanks a lot.
So you mean to use a combination of Pfu and Taq? How about your expanding time? 1kb/ 2minutes or 1kb/minute? I set the expanding time 8minutes for my 6kb fragment, but I got a clear specific band around 2kb. Does it mean that the expanding time is not enough?
I will try the combination of these two polymerase, hopefully it works well:)

There is a standard protocol for longer amplifications.
Make a normal PCR protocol with an apropriate elongation time, then programm it so that each cycle the elongation time is incresased by 5-20 s.
In addition you can use the mix that arwen76 proposed.
It is often called elongase (or similiar) and you can get it from about any polymerase manufacturer.
If you need higher fidelity as obtained from Taq there are also proof reading polymerases around which are usable for long PCRs (e.g. KOD from Novagen).

I just amplified a 2.5 kb fragment from human genomic DNA. It takes a lot of effort to succeed it. i hope my experience will be of assistance to you. at first, i didn't get pure or high quality with BuccalAmp DNA Extraction Kit, because i can't amplify anything from this template. then i used the DNeasy tissue kit from Qiagen and got high quality human genomic DNA. and i believe the polymerase is very important for us. i didn't get anything with Hotstart red Taq from Sigma. but when Expand high fidelity PCR system from Roche was used i got the expected 2.5kb band. the programme is 95 centigrade X2min, then 40 cycles:94 centigradeX40sec, 55 centigradeX40sec, 72 centigradeX3min, the final elongation step 72 centigrade for 7min on BIORAD iCYCLER machine.
my advice is check your genomic DNA quality and select potent polymerase.
good luck!

Hi,

I tried long and hard to obtain my 8 kb band from genomic DNA.
In the end I used DMSO 1-6 µL/50 µL and three different magnesium concentrations 1.6, 1.8 and 2.0 mM.

I used Elongase. happily I obtained the band at 3 µL DMSO/50 µL at 1.8 mM magnesium concentration.

I also ran a gradient PCR running from 65-75 degrees in my case.

I hope this can help!

Cheers,
Martina

Hi,

Roche sells a kit for long template amplification along with an enzyme mix of proofreading & high fidelity polymerase (Expand long template PCR system).

I tried this kit, worked well for me.

Cheers,
Freiberger

PCR protocol are going to vary depending on the G+C content of the region you are amplifying. There are many commerically available "long PCR" enzyme mixes available. Most reply on the high processivity of proofreading enzymes. It is funny, but they are always trying to outdo each other as to which enzyme will produce the longest pcr product.