Simply you change the primers. Change primers length (24-28) to increase the specificity and keep GC content around 50%. Avoid self complementarity. I believe you can solve it easily.

Hi Celia!

Have you checked your DNA? Is the quality ok? Make sure that there is no degradation. Also check the amount of input DNA in each reaction. To much DNA can inhibit the PCR.

I agree to Britta.

Please quantitate your DNA samples. Ideally for a conventional PCR 100-200 ng of DNA gives good PCR amplification. Sometimes even a lesser quantity does the trick.

Also check DNA integrity/quality on 0.8% AGE as mentioned by Briita.

Happy Standardizing Celia!!

I too feel the same as poonam and britta. when you are getting band of your interest in few samples, it means no problem in primers or PCR protocol. Check your DNA concentration and for protein contamination as proteins can highly inhibit your PCR. Make sure same concentration of DNA is used (Note: not the volume of template) in the PCR.

At rare cases there maybe a change in primers, hence after correcting DNA concentration problem seizes please do a primer sequence to check whether you are using the right primers.

Hi Celia,

For optimising I suggest:

Check template quality - is it degraded? How was it purified? If there are inhibitors in the sample then the volume added to the PCR will affect the concentration of inhibitor also added.

Check template quantity - as suggested previously :)

Primers - are they the correct sequence? If you did not design these yourself, check the sequence because many published primers are incorrect. Ensure that there are no SNPs.

The small fragment on the gel is likely to be primer dimers, these will form more readily when there is no target present. The fact that you get more successes when you use more samples could indicate that the ones that fail have poor quality/low quantity target.

Alternatively, it could be that when you use a few samples you aliquot to the edges of the PCR block and in this region the conditions are less suitable than in the body of the block (most instruments have variable temperatures and ramp rate effects across the block). This would  suggest that the PCR conditions are not optimal, in that they are sensitive to the position on the block. What happens if you run triplicates of a sample that has amplified? Do all triplicates amplify? This is a way to test optimisation.

You say that the annealing temperature is 62.6oC but the Tm of the primers is less than this. This means that you are really pushing the annealing. The absolute Ta will depend on the buffer conditions but Iwould have expected to run that a little lower. Maybe try a run at 55oC with some samples that have amplified and some that have not.

Good luck

Hi Celia,

As I see, you have received a lot of wonderful answers :) And this is what I think.

First of all, I think the bands that you observed whose sizes are less than 100 bp, they could be primer dimer, not your amplicons.

Second, with the fact that you had got bands of expected size for some of your samples, your PCR conditions seemed to be optimized already. If not, you would not see those bands. To make sure those are the right bands, you can try to sequence them and double check with the gene bank. If the information of amplified products are not available, just have some faith in the bands that you got. So problem seems to be your sample alone.

- Your samples (not get bands) simply don't contain the gene of interested.

- Your DNAs have degraded (DNA quality). You can check DNA quality by loading them on 0.8% agarose gel. If you see smears, DNA is not good. If you see a band, even light, DNA is fine for PCR.

- Your DNA inputs weren't optimal. You didn't mention anything of how you took care of the input amount of DNA or running a gradient DNA. The amplifications can be affected by amount of template. Too much or too little are not good, it has to be just right or around that  range.

You mentioned, some samples gave amplification and some don't. This can be caused by the ones with bands had just right amount of DNA while the other one didn't. To me, it seems like you extracted the DNA and run PCR with certain volume of extracted DNA. All samples had same input volume which doesn't mean they have same "amount" of DNA because DNA concentration can be varied.

If this is one of the cases, I would:

- take DNA outs, check for quality with the gel,

- using nanodrop or qubit to check for DNA concentration.

- Make standard DNA solutions (for example: 15 ng/uL), this will help you with managing DNA quantity as well as setting up PCR.

- Try to set up PCR reactions with 3 uL of standard DNA solutions (you input DNA is 45ng)

- If you still can see amplified and non-amplified samples, Take one of the non-amplified samples out and run a gradient template.

With standard DNA solution, you can just set up PCR reaction with 5 uL of (template + DI) for template gradient. Set up 7 PCR tubes:

1. Positive control (with a amplified bands sample)

2. Negative control (no DNA, just 5 uL of DI)

3. setting up 5 tubes with amount of standard DNA varied: 1-2-3-4-5 uL of DNA. In each tube, bring to volume of 5 uL with DNA. Finishing this step, we have 5 tubes with 5 uL of template with varied amount of input DNA.

Run the reaction under regular PCR condition. Moreover, having the same input DNA across samples can be used for rough quantification comparison.

I hope this help

Thank you

Thanh Nguyen

Dear Celia,

For God sake if you are performing a simple PCR reaction, so if at the end of the day you get different products on your gel, it most strongly point to the design of your primers. Please make sure the complementarity between your primers is close to zero so inevitably if not you get primers dimers and that is what you see on the gel. Change your primers and also include a positive control to see if you are doing the protocol falwlessly.

Regards,

GCK

Dear Celia

the range of temperatures tested as annealing temperatures appear a bit narrow and high. I usually start with a wide optimisation e.g. between 46 and 66 C then narrow it down in  a second temperature gradient. Also have you looked at your target sequence? I have had a lot of trouble before with a sequence that contained 13 T's in a row, and the only thing which worked was decreasing the extension temperature (not the annealing temperature) to 62 or 60 C; In this case you could opt for a two-step rather than the conventional 3-step PCR protocol.

I also agree with the point made by others that too much input template can be inhibitory, as the DNA will compete for Taq binding. This is the main reason why there is a saturation after 35 or so cycles of  PCR - it is not because the nucleotides or primers are used up, as they are used in large excess.

Quality of DNA is important, as suggested by Thanh

Good luck

FHF

Many thanks to you all!!! It has finally worked: Less DNA and an annealing T of 58ºC