The main factor that affects a PCR reaction is the reaction time of the elongation step. Try to increase the elongation time... It is because the reaction may took a little bit longer, and your amplified chains (products) do not contain the sequence for the second (reverse) primer... that was my problem in one experiment some time ago... Hope this help, good luck!

1-Try two step PCR 2-Use a mastermix from Qiagen or Fermentas (High fidelity, Flash ( two enzyme system+a very good buffer system). Let me know if you need help with the PcR program,. Good luck!

I am using the chemicals from Sigma Aldrich

I think there's a problem with your DNA isolation.Try another method with better performance

I think you need to optimise your reaction mixtures by adding varying concentrations of Mg2+ for example 1, 2, 3 microlitres and then you select the conc that produced the best bands

Since, the composition worked well for another amplifications, but not now means, u need to check out whether your designed primers are good enough or not. Some times, gene specific primers also fail to amplify the gene. Better also try with 20pm conc. primers. I experienced the above two in my gene cloning.

maybe try optimization with different concentrations of Mg2+ and different elongation temperature, if it does not work only for this one gene, than maybe new primers

Mg2+ concentrations may be the problem. Try to add it with your reaction mixture at varying concentrations.

Mg2+ tiration might solve the issue. Try by adding 0.5, 1, 1.5 and 2 microlitres of 25mM MgCl2 to your reaction mix.

what is the GC content of your gene? Do you use any DMSO?

Increase the volume for 50 ul (evaporation always occurs). Precipitate again your template DNA, since there can be always some phenol left. Calculate again the Tm using thee oldest possible method. For every A/T you give 2 degrees, for G/C 4 degrees. 10A/T + 10G/C in your primer means 20 + 40=Tm 60 deg. Hope one of this tricks will help you:)

Did you try optimizating cycle number? also you can try increase the amount of primers

Buy some different primers, try those. if PCR does not work, moving the 3' end of the primer can help.

Also 10 mM dNTPs sounds very high, it is the same as the stock that we use. . Usually we would use about 1/100th of that

I would suggest you to try PCR at different concentrations of template DNA. Try extension time 1 mint and 20 seconds. Melting temp 57 should be enough. Best of luck

You should vary your DNA concentration It should be near about 50ng. Sometimes the high concentration of DNA results in non amplication of desired gene.

I would suggest the following:1- order the PCR master mix, HF flash from Thermofisher(fermentas). 2-make a 1 uM primermix solution ( 1 ul, 10 uM primer F + 1 ul, 10 uM primer R+8 ul dH2O).

12,5 ul PCR mastermix

10 ul prime mix

2,5 ul template

-----------------

25 ul

PcR program:

Step1: 1X

95 C /10 min

Step2: 40X

95/ 10 sec

60C/1.0 min

4 C/hold

If you get primerdimer, add 1 ul dNTPmix (mix 1 ul of 2 mM dNTP with 4 ul dH20) and 1,5 ul template.

good luck

May I know you primers sequence, name of the gene and organism you want to amplify the gene from. Also re-check the primer sequence that you ordered. Next, do you have DNA gel image and not the ratio? You may re-check the DNA concentration, and may increase the amount in PCR reaction? Hopefully, you have taken care of removing completely phenol and chloroform from your purified DNA? Also let me know your PCR cycles condition. For Taq polymerase and for 1.2 Kb size product you should use 1.2-1.5 min elongation time. Final extension should be 10 mins.

Dear Vikash, I have few suggestions for you.

1) check the Tm using NEB Tm calculator. Whatever it comes use Tm of Lower value for both primer.

2) Check the effiecincy of Ploymerase , How much time it takes to amply your gene of interest.

3) If the amplification is not comming then still don't go for higher primer conc. because it may form primer dimer.

4) You can run a 1-2 or higher amount (depending on conc) on 0.8% of gel. Generally people say that genomic DNA is bigger in size it will not run but actually it runs and if everythong is fine then it will be a sharp band. I am telling this because nanodrop may gives false reading because it gives reading for intact DNA and fragmented.

5) Sometimes Phenol content also comes along with DNA while taking the upper layer out during DNA isolation. This interfares with PCR. So during seperation of upper layer don't try to take as much as you can because it permit the phenol content also.

6) addtional MgCl2 and DMSO are final and required in rare cases.

What is the sequence of your primers? Also what polymerase are you using?

Nevermind I see you are using Taq. Taq (at least in my personal experience) does not work well for longer PCR products. Recommend that you switch to Phusion or KOD polymerase. Phusion polymerase has the advantage of a calculator on NEB's webpage. This page re-calculates your Tm and takes into account the salt concentration in your reaction- as these can alter your actual Tm. Also if you are getting multiple bands you may consider checking primer specificity. There is a simple way to do this:

1. Go to: http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?ORGANISM=9606&INPUT_SEQUENCE=NM_001618.3&log$=seqview_box_primer

2. Copy and paste in your template sequence

3. Specify the organism you are amplifying from (as a word of caution if I am amplifying a gene that is cloned into another source - ie: a human gene cloned into E. coli- I put the species name of the genetic background of the host (ie E. coli in the example mentioned above).

4. Based on the Tm you calculated with enter this into the parameters as well (optional)

5. Hit Run

The program will tell you if you are likely to amplify any other genes. Note that you may see the same gene repeated many times and it will think it is non-specific but it is a good test and I have had luck with it.

i think you should optimize your PCR master mix like changing the ratio of taq buffer MgCl2 and dNTPs concentration

Be sure that your primers are unique.

Primers are gene specific

The polymerase is almost almost certainly an issue. Standard Taq does not cope well with amplification of products longer then 1kb and I wouldn't waste anymore time trying to get this to work. Instead I recommend going with one of the long template polymerases such as Phusion, Pfu Ultra etc

Increse the MgCl2 concentration and try to use 40 ng/microlitre DNA 2 microlitre for each reaction. And check annealing at 59 to 62 degrees you might get amplification. If not then certainly there is problem in primers I think. You can go for 55 degree annealing too. Try to do it and ask if any problem re appear

Always run a positive control with a known gene with the same master mix except primers

i am facing the same problem. i am trying to amplify a gene from human gDNA and further want to sequence it. i want to amplify complete CDS region of the gene which is of 1.3 kb. i did not get any amplification. i changed my primer set for 3 times because i was thinking there must be some problem with primes because i used to run a positive control every time and got good amplification every time. i have changed two types of taq but fail. then i changed mgcl2, DNTPs, and template concentration. DNA quantity and integrity id pretty fine. i also played with PCR conditions like extension time and annealing time but again failure. i am stuck. please help. actually i am trying to amplfy two genes having size 1.3kb and 972 bp. i am fail to get amplification of none of them.  

Hi Mohit,

Let me know your PCR cycle condition both for the control and your samples.