long amplicons are dificult to amplify, but requires less cycles. Try to add the taq polymerase after the hot start ( at Cycle 2), at least you are used a enzyme which needed the hot start. Reduce your annealing time to 15s and increase extention time to 3:30 min. Reduce cycles to 25. Good luck. It results improve you PCR , likely by adding 5% glycerol (stock 50%, molecular grade) in the PCR mixture.

Hi, the program you use seems OK but which polymerase do you use? Promega Go Taq?? I would suggest to change the polymerase.

long amplicons are dificult to amplify, but requires less cycles. Try to add the taq polymerase after the hot start ( at Cycle 2), at least you are used a enzyme which needed the hot start. Reduce your annealing time to 15s and increase extention time to 3:30 min. Reduce cycles to 25. Good luck. It results improve you PCR , likely by adding 5% glycerol (stock 50%, molecular grade) in the PCR mixture.

I use the following cycle with many of my PCR and it works like charm for long fragments very well:

95C - 5 min; 20 cycles: 95C-45 sec; 55C-30sec, 72C-3 min; 72C - 10 min.

I use this cycle with Green Go taq from Promega.

Good luck.

What is your template (gDNA, cDNA, plasmid) and how much are you using? You could try increasing or decreasing the amount of template and include a positive control reaction as a quality check.  You could try a different polymerase, some of them are suited for longer products (LAtaq, PlatinumTaq). 

All the suggestions above are very nice. However,  I think the polymerase is making the trouble. I have been using rTaq and Ex-Taq to see thier performance in the amplification of long DNA segments and I found good results while using Ex-Taq. I think you can try your protocol once with Ex-Taq. In addition to that increase the extension time as suggested by Mr. Muralidharan Jayaraman.

Best of luck

I would recommend you to add all the required constituents of PCR separately instead of using PCR mix, and increase in the extension time to 4 min might help you in getting good targeted amplicon.

Hi

increase the time of extension time to 3 min

1. Check DNA template on a gel: to see if they are intact or degraded before use for PCR

2. Double-check your primers: whether or not they are correct

3. Use long-ranged PCR DNA polymerase: LA-Taq > Ex-Taq > Go Taq  (Takara and Promega)

4. Reduce '3-step cycles' first step 94oC from 1:30 min to 15-30 sec, increase extention time a little

5. Add DMSO (~4%) in PCR reaction: to increase efficiency and specificity in case your DNA sequence is G-C rich

6. Make sure PCR machine works properly

1- 94C for 5 min

2- 10 cycle of 94C: 1:30min, anealing at 50C :30s , extetion at 72C : 2:30min

3- 72C for 10 min 

4. 

94C for 5 min

5- 25 cycle of 94C: 1:30min, anealing at 55C :30s , extetion at 72C : 2:30min

6- 72C for 10 min 

7. 4C forever.

I make sure this will well.

Hello Raghdah

An extension step of 2min30 for amplifying a taregt DNA of 2355 bp is definitely inaccurate. The rule-of-the-thumb for PCR amplification was and still is 1 min by 1000 bp to amplify, 2355 bp is higher than 2000 bp, hence 3 min of extension duration.

45 cycles seems to me too much.

Therefore, I dor ecommand you try an old trick (back to the beginning of the PCR some 25 years ago). Make a special PCR program with 42 cycles. The 2 first cycles have an hybridization step at 45°C (yes you correctly read: 45°c, so you force hybridization by being non stringent) and the 40 (or 38) foillowing cycles you use the stringent temperature of  60.5°C. 3 min per exteension step and ... 10 sec at 95°C (it's sufficient to denaturate well any DNA).

Other tricks are to add supplemental chemicals in your PRC amplification reaction, such as trimethylammonium 50 mM or the mysterious but quite efficient Perfect Match PCR Enhancer developed first by Stratagene and now sold by Agilent. I joined the spec file of this compound. Just add 1 unit of PerfectMatch and ... you will see! It costs, if I'm right, about 7 dollars one reaction so it's not cheap but it worths it.

Best regards,

Prof Philippe Urban

For large PCR products you should use an appropriate Enzyme, like the Elongase from Invitrogen (Elongase TM Mix - Invitrogen). As for the program you are using, try to increase the time for the extension step.

Toying with annealing temperature will increase or decrease the specific binding of the primers. Try at the optimal annealing temperature and double or triple the extension time. But if I were you i would use a proper PCR mix for it.

i am using genomic DNA, I will try all of ur suggestion and i hope it will work out for me, thankscall of you.

Hello,

First i think you should reduce cycles number to 35-40 max, because too much cycles could kill your enzyme and your final 10 minutes extension might become useless.

You could try to reduce your annealing temperature to 55 and see what you get.

Increase your extension time to 3 minutes.

What about your template: did you try different concentrations for the PCR?

You can try to change all those changes in 1 PCR and see if you have something.

Sometimes you should only get a different enzyme (like HiFi, cliniscience, it works magic for me). In our lab we do not like ready mixes it is limited when you want to troubleshoot a PCR.

Good luck,

1. I have pasted your 2 primers into Integrated DNA Tchnologies (IDT) OligoA nalyzer 3.1 for primer Tm analysis [ http://www.idtdna.com/calc/analyzer ].

2. The Tm results showed: (1) forward primer= 55.9oC and (2) reverse primer= 56.1oC. You have set the Ta (annealing temperature) = 60.5oC. Although in quit a few PCR reactions, product can be amplified when Ta is set with several degrees higher than Tm (can be tolerated) , I will be cautious and more conservative in setting the Ta for the beginning of the experiment. You may try one PCR reaction with lower Ta to check out whether or not this is a factor. 

Some of the previous comments are accurate and useful. I would advise to test first with the positive control to set the PCR by reduce the time of Ta and try also different concentration of the MgCl2 (Co-activator of the Taq).

there are already good suggestions out, but in case that you still need help, I'd like to share my experience with you.  NEB Phusion enzyme,  95C 45s, 45C 45s, 72C 3min, total 30 cycles, this is useful for almost all the

First, what is the purpose of your amplicon?  Are you subcloning it, or is this only for detection purposes?  If you are subcloning, I would keep your cycle count low and make sure you are using a very high fidelity enzyme. (I am not familiar with the one you are using) to prevent PCR based mutations.

I would second the use of different enzymes.  Phusion has worked very well for me.   Though you could try PFU Turbo as well. 

Also, do you know that your target sequence is wild type?  Are you using primary cells/tissue or are you using a cancer derived cell line?  If you are using cancer cell lines, the sequence may be gone or altered.

Finally, have you blasted your primer sequences?  If you are trying to prime off of a repetitive sequence, there may not be enough primer left for specific amplicons.  If this changing your oligos is not an option, you could try a nested PCR or using a Bacterial Artificial Chromosome as your template.

Moving forward:

1) Get a positive control so you know you can amplify other sequences off of your template.

2)  Lower your cycle count

3) Lower your Ta to 55 - 60 (based on your specific oligos)

4) Add 3% DMSO to your reactions (this may help with secondary structures)

5) Try different enzymes (these will have their own protocols)

6) Try a nested PCR or a BAC based chunk of gDNA

Good luck!

You can Design internal primers also to see the amplified product is correct or not .Usually more than 1.5Kb it is difficult to amplify.

all the best

Hello, what did you do to get better bands? and more intense.

I have the same problem Raghdah Mukhtar

Thanks!

Hi, for getting amplicon around 2500 bp first you have to avoid using master mix. Try to make your own PCR mix by adding a calculated concentration of PCR reaction components, more importantly dNTPs, and Taq polymerases, and primers. For getting good PCR product of 2500 bp, you have to add exact or a little higher concentration of each dNTP for making sure that your reaction mix has a sufficient concentration of each nitrogenous base for getting your desire amplicon for 30 or 35 cycles. Moreover, your PCR protocol looks good, I mean denaturation, annealing and extension time, however increase an extension time a bit to 3 min for each cycle, and 15 min for final extension. Good luck.