no result with pcr. is it the primer?/ - no amplification in pcr even after starting from the beginning (Apr/30/2006 )
dear members
i have been trying to amplify a fragment of the size 2000 bp. i was getting amplification but now i m simply not getting.
in vain i have started all from the beginning ie DNA extraction. still i m not getting the amplification. is it the primer
now please tell me how do i know that the primer is spoilt. yesterday i ran the 2 ul of 25 uM primer in agarose gel electrophoresis but i did not see anything.
please help
i have been trying to amplify a fragment of the size 2000 bp. i was getting amplification but now i m simply not getting.
in vain i have started all from the beginning ie DNA extraction. still i m not getting the amplification. is it the primer
now please tell me how do i know that the primer is spoilt. yesterday i ran the 2 ul of 25 uM primer in agarose gel electrophoresis but i did not see anything.
please help
So it seems there are some problems with your primers.
you might want to try running your primers on acrylamide, they are usually too small to get resolution beyond a faint smear on agarose.
just order up a fresh batch, if it is easy for you to do so...?
I am not too sure if you should be really expecting 2ul of 25mM primers on the gel as I have never done so, but I would go as follows if primer ordering is an issue
1. Confirm that DNA is ok and concentration is detectable on spectrophotometer. I use DNA of approx 200ng/ul conc
2. Make sure there are no extra salts left in the DNA
3. make sure that all reagents you use are in good condition (This you can't test but you know best how they were stored and used meanwhile so you can guess)
4. Primer Tms are close to each other
5. If you are amplofying 2Kb region, keep extention time of 2 minutes at least, mine is 2.30 mins
6. Final extension is about 20 mins
7. I had this issue in the past but it was the annealing temperature that was causing trouble so I increased it to 61 from 58C, when my primer Tm was about 68C or so.
Designing new primers won't work if your reagents or method is faulty.
Hope this helps
i have been trying to amplify a fragment of the size 2000 bp. i was getting amplification but now i m simply not getting.
in vain i have started all from the beginning ie DNA extraction. still i m not getting the amplification. is it the primer
now please tell me how do i know that the primer is spoilt. yesterday i ran the 2 ul of 25 uM primer in agarose gel electrophoresis but i did not see anything.
please help
Do you know if anything in your lab has changed since your expt was working? Try chucking out all of your reagents, including the working dilution of your primers. I mean everything, including the water. Also, test your template DNA on a gel; if it's lousy, there's your problem.
Try to get reagents from a different lab if possible; this will tell you if something is "off" in your lab. If it still fails, you might have a systemic problem - I have had PCR fail because a bad MilliQ filter was installed.
Try not to use MilliQ water if possible, but commercial ultrapure water (DNAsefree) if possible.
The biggest problem whith PCR is you cannot be sure what is going wrong and you have to experiment. But I would buy a new primer. Nowdays they are extremely cheap. (Like $5 each)