I think may be next time with 250ng you may not get band ...because high amount of template DNA also cause inhibition in PCR reaction..I usually took 50 to 100 ng of DNA for my pcr reactions and its works fine for me everytime.

Hi Najla,

it depends of the final volume but you know, 10 ng of DNA is largely enough for genotyping (it represents 1500 human cells). When concentrated, DNA disabble access to enzymes and the first denaturation step could not be enough to get amplification.

fred

Thank you Frederic Lepretre and Sandeep Kumar

when I first designed the primer

I tested it in different concentrations for 7 sample

I got many unspecified bands in 200 ng and a good band in 500 ng

thats why I am using 500

With less concentration than 200 I usually don’t get anything

just as an example, I worked for years doing PCR for sequencing or genotyping and using only 10ng of human DNA in a 10 ul reaction volume. and it worked every time.

so I wonder if your primers are really specific and therefore enough efficient to get amplification. one way to test your primers is to use the NCBI primer blast tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) or the UCSC in silico PCR one (http://genome.ucsc.edu/cgi-bin/hgPcr) just to see if you need to redesign primers. can you tell us more about your work, the species you're working on and the gene you target?

fred

Thanks for the tool Frederic Lepretre

the primer are not the issue I'll try to give you an idea about the work

I'm working on ATRX and the PCR product is around 400 bp (a bit large but needed)

So I know that the primers are okay because when I optimized the conditions I submitted 6 samples for sequencing and they had a good sequence

The condition was ( 500 ng of sample in a total volume 30 ul, 200 gave many unspecified bands and 50 gave nothing)

So after that I want to digest my sample in a restriction enzyme and run an ATRX PCR again

But because the buffer with the restriction enzyme is 10 fold higher in salt concentration than my pcr buffer

I wanted to tested it first without wasting restriction enzyme

So I prepared 3000,4000,5000 ng of a sample in the buffer and took (1/10 the volume to adjust salt conc.) which was 2 ul and did a pcr to 20 ul

only 5000 ng gave me a band ( which correspond to 500ul in the pcr) and that what I expected from previous work

I wanted to know if I can go less than that so from the same 5000ng I made 500, 400 and 250 pcr reactions and this time 250ng gave a band and not 500 or 400!

This is why I'm confused

Thank you so much

Ok, Najla Alogayil , but did you test your DNA before amplification?

just agarose gel with the DNA or a run on an agilent bioanalyzer (or something else as this one) will give you an idea on the degree of degradation.

more DNA are degraded and more it's hard to run molecular biology techniques, when possible.with those needed quantity, I guess your samples are degraded.

for memory, 400 pb amplicon is not very large. I was used to produce 400-500 bp amplicons for sequencing, and designed primers for 12kb amplicons that worked fine.

fred

Frederic Lepretre Thanks a lot for all the suggestions

yes I ran them on gel and it looks of a good quality

I repeated 500ng and 250ng today using four reaction for each just to make sure it’s not a random sampling

and I got a band in all 4 250ng samples and only one faint band in one 500ng

I will update you if I have anything new

appreciate your help

when too much concentrated, each DNA led to poor amplification due to hard access of enzymes and stuffs to the targets.

fred

You can try using DMSO. It helps primer annealing. Sometimes addition of DMSO helps gaining desired band for different template concentration.

What is the age of your stored DNA? If your storage condition is ok then age don't impact much. But lots of freeze thaw cycle impacts on DNA quality.

Thank you Md. Mostafijur Rahman

they are a little old but with good quality

I use them only to test the condition before using the actua sample

I might try DMSO

thanks again

it´s strange what´s happening to you on a PCR that already worked; so we have to figure out that something has changed, you know, degraded primers over freeze thaw cycles is the first thing it came to my head, maybe it´s not.

you can also try touchdown PCR for more primer specificity; I left you an informative link...

https://bitesizebio.com/2203/touchdown-pcr-a-primer-and-some-tips/

Good Luck!

Hi, Najla Alogayil.

In order to evaluate a little bit better your problem, may you tell us the primer sequence and cycling conditions? If not, please, check what is the melting temp of both primers and what is the annealing temp you decided to use and let us know. Ok?

Maybe your annealing temp is not good enough. Have you performed gradient PCR? And what about the amount of GC in the sequence being amplified?

All the best.

Thank you Oscar Recasens and Adriano Costa de Alcantara !

I don't mind sharing the primes, unfortunately I don't have this information right now

The annealing worked well for several reactions and different samples

Now I got a steady amplifications using a 100 ng when I used a buffer with a BSA

I repeated it 3 times and it worked for all of them!

Appreciating all the help

Thanks again