I successfully amplified my samples using Primer set 1 but when I'm amplifying the same samples using different Primer set, the genomic DNA is still stuck in the Wells and only ladder resolves. My primers are new, buffers are fresh, DNA is 50 ng/ul and Primers are 10 pmol/ul. I even tried other PCR settings but nothing changed. No amplification. I used PCR conditions from other articles and still didn't help. Same samples are working well with other primers.
If everything is fine then problem may be with your primer. Please check Tm value (annealing temp.) If it is not experimentally validated, put a gradient PCR. If there is no error with Tm, then check your primer sequence by Primer BLAST and also by insilico PCR (http://www.bioinformatics.org/sms2/pcr_products.html). In primer BLAST it should hit your gene of interest.
There may be a chance for non-working of your reaction mixture so try a positive control.
All the best
Sam
Smita,
If your desired fragment of DNA is not amplified, you must see your primers at the end of the gel. Are your primers there? if no and you can not see any primer at the bottom of gel. it means that DNA is synthesized in large fragments so you have to reduce elongation time. in this case you should also check your primer concentration, sometimes primers are not synthesized properly. If yes, the situation is more complicated. there are several reasons for such a condition including inadequate denaturation time, your annealing temperature is high, your enzyme is inactive or you have some impurities such as some ions that inhibit your enzyme. caution: do not use latex gloves when you are mixing PCR solutions, latex inhibit PCR reaction.
Thank you to all of you for your guidance.
I did a primer blast and got the correct match so my primer sequence is correct.
I do not have any bands at the bottom of the gel. Only my Wells have a glow. I also tried Tm values for 45, 55, 62 degrees. Nothing gave me results. So what can be done next
If well has glow it means problem is with DNA extraction, DNA may have contaminants which may prevent DNA amplification
And if same sample gave you results with other primer set then surely the problem is with primers,
Check different Tm by having gradient PCR and also try the Ist primer set in the same reaction
Better to order a new set of primers and then try it, hope it will solve your problem.
Better to order a new set of primers and then try it, hope it will solve your problem.
As per genomic DNA is glowing in the well is considered it means That your DNA sample contains some impurities in the form of Protein or degraded DNA. It will be better to again extract DNA following the manufacturer instruction strictly.
What type of PCR are you talking about? Phusion PCR or the regular type? I had a similar problem recently. If Tm gradient did not work, order a new set of primers. If you are using phusion PCR, add 0.5uL of MgCl2 (50mM, pre-heated at 65C) to your reaction mixture.
All the best!
Hello Smita
do you still have the problems?
"I successfully amplified my samples using Primer set 1".. can you Show us an Image ? Your image shows as Hanif Ur Rahman most likely a secoundar structures with Proteins or DNA. What Kind of sample is it, meaning what kind of organism?
You only exchange the Primer, right?
You didn't use a new extracted DNA?
try to do 2 times PCR reactions. First PCR product should be purify by using PCR purification kit and use that one and do another PCR again. You might get clear sharp bands you want. If not, your procedure and primers were wrong. Goo luck!!
Hello Smita,
Maybe it's a GC-rich or sencondary structure problems. You can try to add DMSO or a PCR enhancer ( http://www.epibio.com/docs/default-source/protocols/masteramp-10x-pcr-enhancer.pdf?sfvrsn=6 ).
Sometimes it's also the annealing time which is to short, try to increase it (30 sec for example).
Best regards.
I think the problem is in the new primer that used
You must check the primer design of it's primer before using
Thank you so much for your valuable suggestions. My primers are correct according to primer blast. I do get good , clear amplification with another set of primers. Therefore, I have decided to go for a gradient PCR and add DMSO to it. I'll let you know about the outcome 😄
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