PCR no amplification - (Sep/03/2012 )

hii

forward primer - 27nt, Tm-65, GC%-48.1
reverse primer - 28nt, Tm-65.1 GC%-46.4

i have set gradient pcr with annealing temp-60 62 64 and 66

early denaturation- 95 C - 4 min
denaturation - 95- 15s
annealing - 15s
extension- 72- 30s
late extension - 72 - 7 min
35 cycles

expected amplicon size- 1 kb

i did not get any amplification

expert advice needed

There are too many possibilities for anyone to be much use to you without further details, such as:

What polymerase are you using?

What concentration of primers?


How much template did you add?

(perhaps post up the details of your entire master mix?)

i am using taq pol fermentas

0.2micro molar primer

50 ng template

30s extension sounds a bit short for 1 kb with Taq. I would have used 1 min. If the template is a plasmid, I would use substantially less (100x). Are the primers just priming to your template, or do they also encode some 5' sequence?

What does this Primer check (select Task: Primer_Check) tells you about Tm of your primers. Is it similar of different?

http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_ep0401.pdf

Why aren't you reading the manual?

Anneal at 60 oC; reduce the initial denaturation time to 2 min (some of your Taq dies before the first step; anyhow Taq activity decreases over the cycles because of unavoidable denaturation); as phage stated: too much template; I use 5ng/kb plasmid in 75 uL master mix; this means for a plasmid of 5 kb you can have 25 ng plasmid in 75 uL master mix. And as phage also said: 1 min / kb PCR product (as also stated in the manual)

Andreea

I still don't get it why are you all so fixed on the plasmids
I amplified plasmids like five times in my whole life, at maximum, all other hundreds of times it was human gDNA. And for that, 50ng is OK.

Trof on Tue Sep 4 08:18:17 2012 said:

That doesn't mean that is the case for everyone, Trof

A lot of us do a lot of cloning work.

In fact, for me, I would be saying this:

I don't know why everyone is so fixated on genomic DNA, I've NEVER amplified genomic DNA from a human or any other mammal

@ Trof: I keep forgetting that plasmids are not the only source of DNA. I am ashamed in front of you again (also last week I was). But isn't there a rule of thumb like 1 ng/kb DNA in a certain quantity of master mix? I am just asking because I did this PCR from a gDNA like 2-3 years ago and I want to do another one in the future (near future) and I am a bit afraid that I would screw it and I do not have anybody in my lab to ask them about it.
@leelee: I know that you were defending me, but Trof is right: we never know what the people asking here are actually using as a template (note to Janesh Gautam: please let us know next time) and we are just assuming that what we use as template is what everybody else is using and this might be misleading. So Trof is 100% right to make this addition/correction to not mislead the asking person.

Andreea

That's why I specified plasmid dna in my original answer. What counts is the molarity of the primer binding sites. If you tie up all of the primer and enzyme binding to sites on the first round, then you rapidly exhaust dNTPs making single stranded DNA product. The ssDNA never gets a chance to become a template for the reverse primer, since there are so many other competing reverse primer binding sites. With plasmid DNA this can happen with relatively small amounts of template. Even worse is a previous PCR reaction with primer-dimers still present. Genomic DNA presents the opposite problem -- very few binding sites, and you must assure there is sufficient DNA present to allow them to be found and amplified.