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Long distance (16 kb) PCR problem - (Dec/17/2005 )

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Hi ladies and gentlemen:

I am a novice in Long distance PCR, recently I am trying to amplify a fragment as long as 16kb.

about one week ago, I was rather lucky to get a 16kb-long segment, which is confirmed to be the desired fragment. when I diluted the PCR product up to 5000 times, I proceed nested PCR with 4 different paires of primers, nested PCR product shows the long fangment is my desired product.
However, when I repeat my experiments, I cannot get the fragments desired? what a frustrated affairs! Anybody can guide me ? Any sugestion is appreciated!

RxnS Condition as below:

Mg2+ final conc:2.3mM
Template DNA(human genomic DNA): ~240ng
primers mixture final conc: 500nM
DMSO final conc: 7% because of the GC content of the gene I amplified is as high as 60%
dNTPs:200uM
10X buffer:2uL
DyNAzyme EXT DNA polymerase from NEB : 1.1uL
Final Rxn volume:20uL

PCR program:
94C 4min
94C 25sec
60C 30sec 10cycles
70C 13min

94C 20sec
60C 30sec 20cycles
70C 13min+20sec/cycle

70C 5min
4C hold

Upper Primer: 21base
lower primer:18base

I own many thanks to everyone who browse this SOS message.

-Cinba-

Hi, Cinba. Here are some troubleshooting methods for PCR which i quoted from the Tavi's website. Hope it helps.

Make sure all PCR ingredients are taken in the reaction (buffer, template, Taq, etc)
Change the dNTP solution (very sensitive to cycles of thawing and freezing, especially in multiplex PCR)
If you just bought new primers, check for their reliability (bad primer synthesis ?)
Increase primer amount
Increase template amount
Decrease annealing temperature by 6-10º C and check if you get any product. If you don't, check all your PCR ingredients. If you do get products (including unspecific ones) reaction conditions as described above.
Combine some/all of the above


For further information, you can also go to http://info.med.yale.edu/genetics/ward/tavi/Trblesht.html

And one more point, should you check you DNA template by running a gel and see whether the DNA is still intact and not degraded.

Good luck.

-Angel1024-

If this is genomic DNA amplification, I'd suggest that 30 cycles is barely sufficient. Try adding another 5-10 cycles. You also may want to add more enzyme and dNTPs in the middle of this process -- the long extension times may be degrading the reaction components.

-phage434-

Hi Angel1024 and Phage 434:

Thanks for you guide,and your suggestion is very appreciated, I have browsed the Web page you provided and I was provoked and I will Try to increase the cycles, and in the middle of the process I will add some more polymerase and dNTPS, I hope these will works. My merciful God please help you ignorant and bewildering lamb!

-Cinba-

Well, one thing you may do is make sure your extension time is optimized. Start with a generous amount of template (e.g. 10pg of your 16kb template) and do 20 cycles with your primers and different extension times. Products should be visible on gel after 20 cycles, using that amount of template.

-sergechampetier-

QUOTE (sergechampetier @ Dec 20 2005, 12:01 AM)
Well, one thing you may do is make sure your extension time is optimized. Start with a generous amount of template (e.g. 10pg of your 16kb template) and do 20 cycles with your primers and different extension times. Products should be visible on gel after 20 cycles, using that amount of template.



Hi, sergechampetier:

Thanks for your suggestion, according to the DyNAzyme EXT DNA polymerase provider's product manual, extention rate is :1.3~1.5kb/min. my cycles have increased to 35, unfourtunately,nothing even an unspecific band can be seen. more suggetion is appreciated!

-Cinba-

OK, time to get serious. Some other things I'd like to know: How did you purify your DNA. What volume of DNA in what buffer is added to the PCR mix. Are you successful with PCR of shorter fragments with this DNA? A control reaction of a 500-1000 bp fragment would make a lot of sense to optimize things. Those primers look quite short for human genomic DNA. Think about longer primers.

Time to try lowering the DMSO concentration -- 7% seems quite high for 60% GC. Do you know anything about the properties of the sequence you are amplifying? Does it have low GC sections? You definitely need to be doing 35 - 40 cycles. Use a very very dilute amount of your previously successful amplification as a control template for optimizing the PCR reaction. Investigate the effects of annealing temperature on this reaction and choose a good one. Similarly for the DMSO concentration. Consider switching from DMSO to Betaine. Adjust the Mg++ concentration higher in steps of 0.5 mM. For those length primers, I would start with a 55C annealing temperature, or run a gradient PCR.

-phage434-

QUOTE (phage434 @ Dec 20 2005, 11:49 PM)
OK, time to get serious. Some other things I'd like to know: How did you purify your DNA. What volume of DNA in what buffer is added to the PCR mix. Are you successful with PCR of shorter fragments with this DNA? A control reaction of a 500-1000 bp fragment would make a lot of sense to optimize things. Those primers look quite short for human genomic DNA. Think about longer primers.

Time to try lowering the DMSO concentration -- 7% seems quite high for 60% GC. Do you know anything about the properties of the sequence you are amplifying? Does it have low GC sections? You definitely need to be doing 35 - 40 cycles. Use a very very dilute amount of your previously successful amplification as a control template for optimizing the PCR reaction. Investigate the effects of annealing temperature on this reaction and choose a good one. Similarly for the DMSO concentration. Consider switching from DMSO to Betaine. Adjust the Mg++ concentration higher in steps of 0.5 mM. For those length primers, I would start with a 55C annealing temperature, or run a gradient PCR.



Hi phage434:

Thanks for you patient answer and detailed guide!

I have extracted the Human Genomic DNA with SDS-Tris-HCl and Proteinase K according to the protocol on the Molecular Cloning-- A lab manual. At last, Genomic DNA is soluted in PH8.0 TE buffer, the DNA concentration is 80ng/ul.

I strictly do the PCR Rxns due to the Protocol provided by the PCR Rxns Kit(DyNAzyme EXT DNA polymerase, Cat NO.: F-522s) from NEB.

I do succefully Amplify the shorter Fragments with this DNA, the shorter fragments range from 6.8kb to 550bp(respectfully:6.8kb, 4.3kb, 640bp and 550bp ), which resides in different parts of the fragments. I even succefully amplify these fragments with the succefully ampilfied 16kb fragments as template DNA. I adimit that The primers is quite short for genomic DNA, but the Genomic DNA structure is rather difficult, GC content is 60% even the Intron is rich in GC. Now I will design longer primers. How long the primers do you think are appropriate for this long segment Amplification?

7% DMSO may be too high, Rather unbelievably, when I suucefully amplify the 16kb fagments , DMSO final conc is 7%.

I has diluted the previously succeful PCR product as template for 5000 times, but I get nothing even an unspecific bands.

I will try a lower annealing Temp, higher Mg++ conc.

-Cinba-

Isn't an elongation temperature of 70°C also a bit high? The longer your polymerase is at a higher temperature, the more activity it loses.

If you leave out the introns how big is your region? (Just to know that if you would be able to succesfully create cDNA, you could optimise your reaction on a shorter region).

-vairus-

QUOTE (vairus @ Dec 22 2005, 07:02 PM)
Isn't an elongation temperature of 70°C also a bit high? The longer your polymerase is at a higher temperature, the more activity it loses.

If you leave out the introns how big is your region? (Just to know that if you would be able to succesfully create cDNA, you could optimise your reaction on a shorter region).


Hi Varius:

Thanks for your guide and suggestion. well I will try lower Extension Temperature.


According to the amplification Protocol with the PCR kit, the extension temperature is 70°C , and 20kb lambda control DNA fragment is succefully amplified extending at 70°C. The polymerase is rather hot endurable, which has a half life of 3.5hr at 96°C, besides it can tolerates high DMSO concentration up to 10%.

The coding region of my desired gene(Human COL1A1)is 4.5kb, and the full length of the spliced Transcript is 6.7kb with 67% GC content. About 2 months ago, I have sought help about this difficult mRNA RT-PCR in this forum,and I still remember you have answered my questions. I even tried SuperscriptII Reverse Transcriptase from Life Technoloy, Gibeco at 50°C to reverse transcribe the mRNA. but I get nothing when I use the DyNAzyme EXT DNA polymerase to amplify the cDNA first strand. So I try its genomic DNA.

More suugestion is suggested. and I need your help! please give me more guide.

Many thanks to you!

-Cinba-

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