Did you check the cycling? Longer amplicons needs longer extension time. Its simple and kind of obvious, but sometimes we forget these simple things. So 1kb needs 1min and so on. And a 4kb amplicon really isn't something that a polimerase can extend easily, of course a smaller amplicon would be much more easily (and eficiently) amplificated. Hope I could help. Luiza.

If you want to try a nested PCR, do the first PCR with primers that flank the region of interest by ~50 bp and then move in from there with your specific primer set. Touchdown or step up thermocycling conditions may also help you get the product of interest.

If you are using EtBr to detect your bands, exposure to UV while cutting the band out of the gel can damage it.

Did you check the cycling? Longer amplicons needs longer extension time. Its simple and kind of obvious, but sometimes we forget these simple things. So 1kb needs 1min and so on. And a 4kb amplicon really isn't something that a polimerase can extend easily, of course a smaller amplicon would be much more easily (and eficiently) amplificated. Hope I could help. Luiza.

Hi,

try to order a pair o primer that will give You a shorter product (difference in size 50 to 100bp should be enough) - and use them in the second PCR. That should help. You can also try some "ready to go" mixes, usually they contain a lot of enhancers and so on - that should help with inhibitors (for example KAPA is producing nice kits), but I would start with primers.

good luck

M

One more thing, what was the extraction method ? Maybe try to cut out the band You are interested in, place it in tube, add some ddH2O and incubate a few hours, use a solution to set up a PCR - it's simple and works nicely (even with a really small amount of material)

I have done nested PCR on cDNA (but not genomic DNA - so dealing with much smaller product sizes) and confirm what Cynthe said re. design of primers - this is crucial. In addition each set of primers needs to be optimised seperately re. temp and MgCl2 etc. I did not clean up the first round product but used it straight in the inner reaction. Also you could increase the number of cycles of the inner PCR reaction and use a bit more.

Not sure if that helps but good luck!

If you do a nested PCR there is no need to purified form the gel. But if you need so,you ca use a melting agarose.cut the fragmente from the gel and purifie whith colun.

Technically you should be able to repeat PCR using the same set of primers and that sometimes works for me, but to design another inner pair of primer should be your next step. The gel purification process may screw up the ends of your PCR product or add comtaminants/PCR inhibitors to your template. From my experience Phusion (compared to Taq) will give you the product with minimal optimization if you use a correct annealing temperature.

There are some points:

1.- Did you eliminate all the alcohol when you gel extract the PCR product???...

2.- Did you resuspend the product in water or buffer???

3.- Check the extension times and try to amplify the nested product with a dfferent enzyme

Good luck!!! : )

You may not use gel extraction or nesting primers because your initial primers are critical. First, re-design the primers to make sure to reduce the primer dimers. Second, optimise the PCR conditions using temperture and Mg2+ gradient, which will give you the best annealing temperature and concentration of Mg2+. If you still see the smaller bands, then use the touch-down method to make sure only to get the single band (your target). Don't use too much template (initially 1-10ng will be enough). If still see the smaller band, dilute PCR product 1:1000, and re-run PCR using the same condition for three times until you see no smaller bands. Good luck!

Conventional polymerases in the first amplificationas do not give a performance greater than 2KB, I would recommend the Pfu turbo. Interfering bands may be due to "hot start" problems for which you can check your anealling protocol and use of enzyme that eliminates the problem as Platinum Taq.

Reviews the purification of your amplified such as lo says Arturo above.

Success!

Hi, I'm not sure the contamination here is of problem, unless you work with other DNA source (human or rat) in addition to mouse. Everybody here has given great advices; one more thing to add is check the GC content. higher GC (60% or higher) will affect DNA structure, inhibit PCR reaction or generate different amplicons. One way to bypass that and to increase yield is to use either DMSO or 7-aza; check also for palindromic sequences. I don't know why you need that sequence but a second option, if you need a renewable source, is to clone the sequence.

Hi,

i think you should increase the amount of dNTPs.or try to optimize with different PCR mixture..I used PCR kit from KOD novagen to amplify bigger amplicon up to 9.5 kb and i only can get a good result with this kit.quite expensive but still affordable to amplify bigger fragment of PCR. thank you

you can use lower amount of template for PCR or If you have just one band in your first PCR, you can dilute the PCR product(1:100 or 1:50) and directly use it as template for the second PCR (without running on the gel and gel extraction)

Hi,

I suggest you, not to go with nested PCR . Instead of that you can double amplify directly once again the pcr product of first pcr run without going for purification. provided that there will be no any nonspecific band. If nonspecific bands are there in first pcr product then first you need to standardize the annealing temperature of primers your using currently to amplify the 4kb fragment. Check the concentration of pcr product of second pcr run on the gel. IF it is more enough with specific band then third time you keep the bulk pcr using the pcr product of second pcr run. Then check the concentration of bulk pcr product on agarose gel if it is more enough then go for the final pcr product purification or gel extraction (if non specific bands are appearing). U can also use the xt5 ta polymerase enz. to amplify such long pcr amplicon size. Also u can check for the time of denaturation, annealing and extension.

If above strategy doesn't work then follow the nested pcr. Your first primer pair should be out the entire gene maybe 50 bp away from both ends of gene and it should not amplify the nonspecific target and second time u can use targeted pcr primers to amplify 4 kb fragment. I Hope u may have worked out the pcr reaction composition .

You might also use the band-stab method, just isolate the band of interest but don't extract the DNA from it. Simply stab with a tip in the gel-slice and place the tip in the PCR solution, just pipet this up and down a couple of times, this way you have added the template.

Hi,

as you were able to amplify in the first round when you used a more complex template, why should you not be able to amplify in the second round with a gel excisted band?

Most likely you overexposed the DNA in the gel to UV light. Try to only very briefely expose the gel to UV light. In most cases this will help you get the long product.

Of course primer design, Mg, Tm and use of a proof reading polymerase will determine if you amplify. But if you have crosslinked or damaged the nucleotides in between you still will not get a product. And the longer the product the more likely it is that such UV damage will happen.

best

Alexander

Hello guy,

I have worked a lot with large fragments. My experience says that it is always complicated.

1) Nested PCR is a solution you can try.

If you do that and if your inital primers are critical, you can first design a first couple of primers larger than the primers for the second PCR, with the first couple containing the sequence of the second, but being longuer in 5' end.

2) Try different polymerases (I know it is expensive), but from one enzyme to another you can be sure to have completely different results. Try to use a PCR for large fragment, a high fidelity , a hot start and a basic ensyme, and look at the difference of intensity and specificity. Do not hesitate in increasing the cycle number to 40-45.

3) If it still appears complicated to succeed, try to clone the product of first amplification, directly into TOPO XL vector.

Often, it is the only way to get your long fragment and to increase the yield by simple miniprep and subsequent PCR on purified plasmid.

Good luck.

TRy diluting the product of the first round of PCR and/or touch down PCR.

Well, first i´ll try to improve the extraction step (see other commercial kits, p.e. qiagen. Second i`ll try to add a few cycles to your PCR program. Finally i`d think in perform a nested or seminested PCR (more probably contamination)

Hi

Are you certain there are no chaotropes like guanidinium in the extract? This will stop a PCR in its tracks every time and is a surprisingly common issue. When extracting, keep things as simple as possible, many commercial kits leave unhelpful residues, in my experience.

Before you try any additional PCR, try a simple Southern to make sure that your Oligos recognize the sequences in the BAC DNA. Once you have established that your oligos are authentic, use PCR kit from Roche, for long amplification product. Generally using a short cut PCR often results in unusable PCR products. During hybridization of your Oligos, you could establish the best conditions for annealing. You may require to define the melting temperature for the best results. It is possible that the 3' end sequences of your Oligos are not right for the elongation, which often happen with commercial Oligos and is ditrimental for a perfect PCR amplification.

One option is to run your first round amplicon in a gel, if you have non specific bands try to take a tiny piece from the right band by a needle and use as a template using thesame parameter you used,hope you eliminate non specific bands without going to gel extraction, otherwise perform nested PCR using smaller amount of template from round one.

I'd follow the simplist method of troubleshooting first and then move on from there should they not work. Optimize your pcr reaction as previously mentioned, that is usually the first step in cleaning up non-specific amplification. If that doesn't get rid of them, I'd try to band stab the amplicon and re-amp using the stab as template...if that doesn't work, try switching enzymes (if you're able), you'd be surprised how differently polymerases behave using identical conditions. If all that doesn't work, you can go with nested or semi-nested to increase specificity.

You can try CloneJET™ PCR Cloning Kit...

Thanks guys!

To address some of the things mentioned here, the nanodrop data of the gel extraction is surprisingly clean, and I do get a ton of smaller products, so it doesn't seem as if inhibitors are the issue, although it could be a low enough concentration that it only affects the long product.

I've been trying several different polymerases, and it appears that accuprime is more specific, but phusion is more processive, I'm contemplating actually mixing the two :P

The needle pricking the gel instead of a gel extract is a very interesting idea, perhaps my gel extraction is too much dna? I'll try that since its quick and easy.

I've been using touchdown pcr primarily, but now I'm thinking that I may want to use a higher standard pcr program. My reasoning is that while TD favors the higher TM primer-product pair, at the lower stages, the smaller amplicons may outcompete by sheer efficiency, since there is nearly an order of magnitude difference in size.

And yep, I think my last stop will be nested pcrs, unfortunately it means I need to remake quite a few primers if that ends up being the main solution.

This problem is just mind boggling, it makes me think pcr does not work like we think it does :D

Thanks all for the help!

What may be most surprising about your situation is that you don't get a large amount of clean specific product directly from the BAC template PCR. If the BAC DNA is reasonably pure, there should be a very high concentration of specific template--only 10-100-fold lower than a purified 4kb segment (as opposed to ~1 million fold lower in mouse genomic DNA). In theory, it should take much fewer than 30 cycles of amplification from the BAC to get a good yield and the possibilities for non-specific primer binding are also much less. So, If you are not getting a good product yield from the BAC template, I think that you should focus on optimizing that PCR reaction and completely avoid the need for a second round or nested reaction. One possibility that you should consider is that the "product" you are seeing so far in the BAC PCR is NOT the correct segment. Is it exactly the right size? Can you cut it with predicted restriction enzymes? It's possible that the large fragment product you purify is an "illegitimate concatenate" of smaller products--and thus has internal sites matching the primer sequences---and this would explain why you get only much smaller fragment products when you use it as template.

It's possible to PCR long fragments from genomic DNA and BAC DNA should be easier. I've been able to do 10 Kbp fragments pretty rountinely. I use longer primers (22-24 bp) with a higher melting temperature (around 62 degrees), , and minimum amounts of template to reduce nonspecific amplification. I do ~1 min/Kb for extension times and also time extension on the PCR and 40 cycles. I've used the Roche Long Template, Accutaq from Sigma, and Invitrogen pfu polymerases. Then, if you get a single band, you can sequence directly. I often use an internal primer for the sequencing, so contaminating PCR products don't mess it up. I have great difficulty cloning long PCR products - if anyone has any suggestions, I'd appreciate it.

HI,

I would suggest to use a kit for the gel extraction that allow to elute in a small volume, so that the ratio of product to contaminant of the GE is low and to estimate the amount on gel against a ladder and not in the nanodrop. If it is not sufficient I would do a SDS, phenol extraction followed by ethanol precipitation so that you can resuspend your PCR product in a TE buffer (10 mMTris, 0.01m M EDTA), to proceed with the second PCR. To increase the specificity of the PCR do for the second PCR a nested one.

I agree with most comments on the issue. You should not expose you band to UV or be quick to cut it. Use a proofreading polymerase with strand displacement capacity. Pfx from Invitrogene is relatively cheap. Also try to put some additives such as 1.5 M - 2 M Betaine in the reaction mixture. I also suggest you UV for 5 min all the reaction mixtures except the template and the polymerase so you get rid of any contamination.

UV will cross link your DNA and block PCR of the template

You should avoid or keep UV to a minimum. Also check the UV wavelength some are more damaging than others

About UV - use 360-365 nm to excise the band. Many light boxes are 302 nm, which will destroy your DNA within a minute.

Try using fresh reagents for long PCR, too. I've been struggling with a couple 8-kb amplicons for a week...switched to a new stock of dNTPs and it worked first try!

For cloning 8-10 kb amplicons, especially if they contain secondary structure, I highly recommend the linear pJAZZ vector (Lucigen Corp., Middleton, WI). I've never had it fail.

[Disclaimer: I work for Lucigen. Nonetheless, the pJAZZ vector is unbeatable for cloning difficult templates.]

@David Barker

Thanks for the reality check, ever since I programmed by hand a touch-down program onto the pcr machine, I've stopped really thinking about it.

I think the first thing I will do is do regular PCR with a constant Tm as I am fairly confident in the bac prep purity and my primer design.

Also, to all of those posting about the UV light damage, you are preaching to the choir :D I forgot to mention it, but because of how much gel extractions we do, I built a blue/green LED box, which illuminates EtBr (green) and GelStar (blue) without damaging DNA. Also lets you cut without goggles on :P

Thanks guys, it'll be a while though before I post an update on the actual results because I'm swamped with a revision deadline. However, I am pretty optimistic (o.O what a new feeling) that these new tricks will solve my problem!

'Dirty' water is the leading cause of non-working and/or messy PCR - use top grade ultrapure water :-)

Re: UV cross-linking.... Mind you, if you're quick at excising the required band under UV there is unlikely to be enough cross-linking to totally eradicate PCR re-amplification. Trust me, I checked this many years ago when using pre-PCR UV 'zapping' (tech term!) to eradicate stray human DNA from PCR reagents... in the days when you made up your own PCR buffers etc from scratch (oh those halcyon days!).

For the record, diluting your first round PCR product might well improve second round amplification - try doubling dilutions or orders of magnitude dilution - it dilutes out any inhibitors and other PCR 'rubbish'. Again, trust me, I speak from expreience having done this with difficult templates.

Best of luck with the revisoin and exams :-)

I have used nested PCR's for amplifying diddlysquat DNA from archival tissues. Match those annealing temps as best you can when you design your primers (and you may want an overhang although we didn't need overhang).

Interesting

Try using a blue light (not UV) transilluminator for band excision.