I can see a couple of things that might affect PCR, lets have a look and see if we can sort this out.

10pM seems a bit low for a primer solution, giving you about 500fM in the final PCR. I would recommend increasing this to about 10µM, which would give you about 500nM in the final PCR. Primers are often the limiting factor in terms of PCR yield, as they are used up in every cycle and oce they run out the reaction cannot continue.

The extension duration looks ok, but you might want to increase it a bit, as the High-Fidelity polymerases tend to be a bit slower than Taq, which is usually quoted as 1000 bases/min, so maybe increase this to 5 min. Don't worry about degrading the template/polymerase, as 72°C should not affect them too much in a PCR mastermix.

Rather than increasing, I would probably decrease the amount of template you are adding. In your first experiment, you have about 40,000 copies of the genomic template DNA and in your second this increases to 94,000 genome copies. The risk here is that there is a large amount of other DNA present that can competatively bind to your primers (even though the binding is very weak because the base pairs don't match, there is alot of it) and reduce their chances of being used to prime your polymerase extension. I would reduce you input DNA amount to about 5,000 genome copies (approximately 4 ng/reaction, dilute 5µL of your stock DNA in 195µL of nuclease free water and use 5µL of this per PCR).

I am guessing that you are using 30 cycles as you want to reduce the risk of introducing mutations into the sequence so you can use it for ligations or something similar. Yes, this can be a problem, but it is more important to have a higher yield, so if the above steps didn't work, increase your number of cycles to 35. Every cycle that you run will give you approximately double the amount of PCR product, so even a modest increase in cycle number can improve yield dramatically.

If none of that improves your yield, try digesting your genomic DNA template with a restriction enzyme that does not cut inside your PCR product. If you get to this, let us know and I will help you pick one.

I can see a couple of things that might affect PCR, lets have a look and see if we can sort this out.

10pM seems a bit low for a primer solution, giving you about 500fM in the final PCR. I would recommend increasing this to about 10µM, which would give you about 500nM in the final PCR. Primers are often the limiting factor in terms of PCR yield, as they are used up in every cycle and oce they run out the reaction cannot continue.

The extension duration looks ok, but you might want to increase it a bit, as the High-Fidelity polymerases tend to be a bit slower than Taq, which is usually quoted as 1000 bases/min, so maybe increase this to 5 min. Don't worry about degrading the template/polymerase, as 72°C should not affect them too much in a PCR mastermix.

Rather than increasing, I would probably decrease the amount of template you are adding. In your first experiment, you have about 40,000 copies of the genomic template DNA and in your second this increases to 94,000 genome copies. The risk here is that there is a large amount of other DNA present that can competatively bind to your primers (even though the binding is very weak because the base pairs don't match, there is alot of it) and reduce their chances of being used to prime your polymerase extension. I would reduce you input DNA amount to about 5,000 genome copies (approximately 4 ng/reaction, dilute 5µL of your stock DNA in 195µL of nuclease free water and use 5µL of this per PCR).

I am guessing that you are using 30 cycles as you want to reduce the risk of introducing mutations into the sequence so you can use it for ligations or something similar. Yes, this can be a problem, but it is more important to have a higher yield, so if the above steps didn't work, increase your number of cycles to 35. Every cycle that you run will give you approximately double the amount of PCR product, so even a modest increase in cycle number can improve yield dramatically.

If none of that improves your yield, try digesting your genomic DNA template with a restriction enzyme that does not cut inside your PCR product. If you get to this, let us know and I will help you pick one.

The fact that increased template amount resulted in no bands, may suggest that the template is not clean enough. I would stick to 3 ul of DNA. I don't know what is the concentration of your polymerase but at the extension step of 3:30 in a 50 ul reaction, 0.6 ul of the enzyme seems not enough. Next time I would use the same conditions but with 5 additional cycles and double the amount of polymerase.

Nested PCR might be a simple and clever method.

- Did you check the GC content of your target region?

- > 3Kb is a relatively long amplicon for a High-Fidelity enzyme, maybe you should shift to a higher processivity enzyme (Long Template PCR Systems). However, before shifting to a different enzyme I would give a nested PCR a try, as suggested by Hirokazu.

X Jacob: imho primer concentration is ok (even slightly more concentrated than usual). Please, note that primers are reported in picomoles (pm), not picomolar concentration (pM), so 10 pm = 1 microliter of 10 micromolar primers (i.e. 10microM = 10 micromoles/liter = 10pm/microliter).

Check you annealing temperature; it appears to be too high. Try a gradient PCR or reduce the annealing temperature by 3 C and try the PCR once again. Another thing that can be done is to gel purify the specific band that you have obtained and re-amplify it.

I think that your problem wil be resolved by:

- Gradient PCR 50-65

- 35 PCR cycle

If good and specific yield was not obtained yet, Use the best yield conditions with different elongation times. add some enzyme.

I

I think increase dNTP concentration.

You will have to standardize and find a best annealing temperature for your product.

As Vikas Jain explained,Go for gradient PCR.

You have a large PCR product, so 30 s is not enough for annealing. Test your  annealing time with  1:30 and 2:00. Also your primer concentrations  are too low. If this is not working just let me know. I can give you more suggestions. 

10pM is definitely low for your primer concentration.  Like others have suggested, up it to 10uM.  Also, perhaps try increasing your annealing temp to 58-59C.  That is where I standardly start with a new primers.

I would say your primer concentration is far too low and to try a more traditional concentration of around 10uM (the stock volume of primer).

I would also strongly suggest a much lower amount of template you really do not need more than 100ng of template. Depending on the abundance of your gene of interest you can go into the pico or even femto concentration range.

I personally use no more than 5ng of template per reaction and would suggest starting there, possibly trying a concentration gradient from 1-50 (1, 5, 10, 20, 30, 40, 50 etc), before changing the PCR conditions or changing the PCR technique its self (if it has previously worked). 

You SHOULD see a much better result using lower template in the initial reaction. Hope this helps!!

Dear all,

Mohammad reported primer  as pm (picomoles = total amount) not pM (picomolar = concentration) so (unless it was a typo and he meant 10pM): 10 pm = 1 microliter of 10 micromolar primers (i.e. 10microM = 10 micromoles/liter = 10pm/microliter). Therefore the concentration of both primers is correct.

Let me know your opinion.

Well in that case it is more than likely the template concentration being too high. Therefore I would say default to the second part of my original answer.

Hope this helps!!!

I would definitely concur with @Rocco Piazza, if the primer values are amounts in pm rather than concentrations in pM then the concentrations used are fine.

I would try to decrease the temperature of the elongation to 68 degrees. In the case of products longer that 3kb I usually use 68 and in 3 cases on 5, it works.

Please check stock concentrations of cour PCR-Mix components (see Primer etc.).

are you using specific primer r random primer?  check the target DNA too, Maximum error happens with primer missmatch ... If you used specific concentrate on Mgcl2

Template is too high - 0.3 - 1 ul at a concentration of 100 ng/reaction will be enough.

Primer - 0.3 - 0.5 at 10 pm or micro mole will be enough.

Put a gradient  pcr to check the annealing temperature.

clearly your DNA  poisons the reaction, so use less or clean it up. check this primer concentration thing, you might always increase it anyway. Also consider nested PCR, using a second pair of primers more internal to your fragment.

I'm used this formula if you want to use it , but I think the problem for you the annealing temperature,so I recommend using 54-56 C

10X High Fidelity PCR buffer with 15 mM MgCl2……10ul

dNTP Mix (10mM)……………………………….……..1 ul

Forward primer (20pm)……………………..…….…...2.5 ul

Revers primer (20pm)…………………………...……..2.5 ul

Template DNA (46ngr/1ul)……………………………….2ul (138 ngr/50ul reaction)

High Fidelity PCR Enzyme Mix……………………..…0.5 ul

Water, nuclease-free……………………………….….32.5 ul

I recommend you to add DMSO to your reaction mixture. That‘s a useful way to get good PCR product. At the same time, your DNA template  added too high, decrease it a little.

Try again ( by using 3µl), however, increasing  to 40 cycles.

Couple of things

First I agree that if your primer stock conc is 10 pM then you need to increase this as suggested

Second, you are starting with genomic DNA as a template. This is difficult to denature initially so I would increase the initial denaturation temp to 97-98C for 3 min. I know the enzyme may not be happy but it will work better. Subsequent denaturation can be done at 95C/20sec. Adding DMSO as suggested will also help but it may inhibit your enzyme activity. Another idea would be to try a system that incorporates a high GC content buffer and try that. I use kappa High Fidelity Polymerase from kappa biosystems and it works exceptionally well. Alternate would be the Q5 from NEB. I have used both in screening for bands of 4.4kb and 6kb and gotten great results

You say on the second day you simply upped the amount of genomic DNA to try to increase your specific product. If your initial primer conc was too low then this would be counterproductive. Genomic DNA tends to be far more hungry for primers than standard cDNA or plasmid DNA.

What I would do is take an aliquot of your first PCR reaction and dilute it 1:50. Then use 1-2ul of this in a nested PCR reaction to generate your band (using a correct primer concentration). The proofreaders nowadays are quite efficient and their processivity is usually 1000-1500bp/min or better so you can go up to 35 cycles with no issues concerning mutatons

There are some issue that are to be taken care. First, the primer concentration that you are using is very low you can increase it up to 20 pmol/ul. Second, you can add 5 ul of 10mM dNTPs mix (for 50 ul reaction mix) so that final concentration remains 0.25mM of each of the dNTPS which is very optimum and add 3.5 U of enzyme since your fragment size is longer than 3 Kb. Third, be careful while mixing primers, all the reaction mix should be kept in ice because Highfidely, enzyme has proof reading i.e. 5'-3' exonuclease activity. If you keep the reaction mix at room temperature the enzyme may chop up the primers. Fourth, replace the aliquot  the nuclease free water with a fresh one. sometimes, contamination in water might inhibit PCR.

               Besides, You may also add 1% DMSO which helps PCR for difficult templates and increase extension temperature to 4 min since, highfidelity enzymes acts slower in extension than Taq. You can also increase the cycles to 35, which can improve the yield of the PCR. Finally to ensure that all the components are working correctly, include a positive control in the reaction. If you get the amplicon let us know.

I agrees with previous issues reported, the main problem is primer concentration or how you dissolved your primers initially. Chick also, activity of the enzymes as frequent freezing and thawing affect its working

I agree, primer concentration seems low. Any way, have you test these primers previously with Taq? Have you perform a gradient PCR in order to know the best annealling temperature?

If all the suggestions you have received will fail...

Try to decrease also dna concentration. You could might have inhibitors in you Dna. 

One solution could be to dilute them till they do not interfere anymore, but stil maintain Dna of course.

Try to perform PCR with  1:1, 1,:2, 1:5, 1:10, 1:20, 1:50  dilutions of  the dna quantity  used in your first pcr.

I do agree with the above suggestions. I recommend you to increase your denaturation time from 30s to 1min, as your DNA may be taking more time for separation.

The above researcher are correct, that the primer concentration is too low to amplify the large fragment. Atleast used 10um as primer conentration and your thermocycler condition looks perfect. If the problem still remains the same use Roche expand long template pcr system or you can try this system thermocycler condition once, this definitely work becoz i had amplify upto 8kb of fragment with this kit. https://cssportal.roche.com/LFR_PublicDocs/ras/11681834001_en_18.pdf 

I agree with the above suggestions.

The amount of template DNA you use is also quite high, you could try to decrease this amount (use about 0.5-0.75% of the amount in the first reaction).

Also addition of DMSO (1-10%, I usually try 5% as a first test) in your reaction could improve your results.

Have you tried changing the annealing temperature? Maybe you should try to increase (or decrease, but you seem to have a quite low annealing temperature already) the temperature for the annealing.

If you had a weak band to start with, I'd go back to your original protocol, and increase the number of cycles

I think it is too much DNA (322 ngr/50ul reaction) for such oligos' concentration. I would increase the number of cyclesto 35 or 40 if you want a strong band (instead of the DNA). Unless I have to excise band, I do PCR reactions of 10 uL (to save reagents), with the oligo concentration that you have indicated and around 75 ng/uL of template (I add only 1 uL of it even for a 50 uL fnal volume). Bests

May be you are taking too much DNA. for 50 ul reaction. so you take around 100ng DNA and 2 ul of (5 micro molar) of each primer, 4 ul (10 mM) dNTPS. Go for 42 cycles where keep annealing for 1 minute at 55 0C. You will have clear bands.

All the best

I never go up to 40 cycles in PCR... With too much cycles you amplify water.

depends on starting template quantity, and every other condition in the reaction... besides, you're going to sequence after, you'll soon know if you're "amplifying water"

if after trying the previous suggestions, still does not work, I would also check the MgCl2 concentration.  Some PCRs do not work well with 1.5 mM,so  try increasing it to 2 or 2.5 mM of MgCl2, and  in the worse of the cases do a gradient of annealing temperature for the primers too.

Probably too much DNA, or other inhibitors are present in your sample. DNA is a very strong inhibitor of PCR. Try using your sample in 1:10,  1:100 and 1:1000 dillution and if you then see bands your problem is reduced to inhibition. 

Hi Mohammad,

Did you fix your problem yet? if no please let me know and I can give you a few other tips to solve it.

First check whether you have added all reagents properly. 

I would like to know what was the buffer in which you have dissolved your gDNA since when your 3ul reaction is working and 7ul reaction not working it gives an indication of PCR inhibition in your experiment. If you dissolve it in NaOH clearly the pH of your reaction buffer seems to be compromised.

Note: you will not receive a drastic change in PCR amplicon amount by just using 2 times more template concentration.

I agree mostly to the excellent reply of Jacob, but would suggest to increase the extension time up to 2.5 or 3 minutes. Your PCR system uses a slower Taq with proof reading function and in my experience you should use approx 1min / 1000bp with this particular kit. Not spending this time may reduce not only amplification efficacy but also proof reading function.

Reducing the target DNA concentration - as already has been suggested by several others - would be my second suggestion, too. 

The last suggestion is a question, if you know that your primers are truly binding to the target DNA? Theory is not always working, as we all know  ;) Your description sounds a bit as if you do not have a positive control yet. If this is the case, you could help yourself by ordering an 50bp oligo with the primer sequences at its ends (this oligo would be used as a target in your PCR and received amplicons can be further used as template for positive control in the future). This gives you the information, if the PCR was adequately running even if all samples could not be amplified.

might be product of pcr can be used to amplify again and get stronger band

Mohammed,

You are using a high conc. of DNA, primer DNTP. Use 1ul of DNA, 1UL OF primer, 1ul of DNTP, 1ul enzyme for 50ul reaction. This u try definitely you will get

Dear shayesteh

I think you should do one or two or more of below options:

1) Use a high concentrations of Mgcl2 (Best option)

2) increase your PCR cycles to 35

3) may be you have an inhibitor in DNA template (DNA must be diluted)

4) change the PCR set completely

I think you use a good amount of dna. In eukaryotic cells, recommanded amount for PCR amplification is 100 ng. Make an horizontal gradient temperature coupled with a vertical gradient MgCl2.

Dear shayesteh,

For your PCR that you are optimizing, I suggest:

Good luck!

Ahmad

Dear shayesteh,,

I think the annealing temperature is critical, which is about 3 degree higher than your primers Tm. You can run the gradient for the annealing to find optimized temperature for it.

Good Luck

Weiping

Have got solution ? if yes post it. Hope so optimization of Mgcl2 will give better result

@Mathieu Dondelinger--What do you mean by "horizontal gradient temperature coupled with a vertical gradient MgCl2"? 

For example : try temperature 50 to 60 °C (horizontal gradient in the thermocycler : column 1 : 50 ; column 2 : 52 ; column 3 : 54 etc. ). For each sample in each temperature, try increasing MgCl2 concentrations (vertical gradient : lane 1 : 1,5 mM ; lane 2 : 1,75 mM ; lane 3 : 2mM ; lane 4 : 2,25 mM ; lane 5 : 2,5 mM). Concentrations may vary depending the enzyme AND the buffer used. So try different polymerase and, if possible, different buffer (buffer with KCl will stabilize primer annealing and buffer with (NH4)2SO4 will destabilize mismatched primer-template). Taq with KCl usualy  goes with MgCl2 1,5 ± 0,25 mM; Taq with (NH4)2SO4 usualy goes with MgCl2 2 ± 0,5. Pfu 2 mM MgCl2 is recommanded. Do not forget to increase MgCl2 concentration if your template DNA  is diluted in a buffer with EDTA. If you do not succeed amplification, watch at your primers (re-order if you are sure of sequences, it is possible an error occured during primer production) and your template dna (purity and presence of pcr inhibitors (presence of ethanol from the washing step - humic and fumic acids for environmental samples). I hope this is complete ^^. Good luck!