15 replies to this topic

[Janesh Gautam]

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

[leelee]

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?)

[Janesh Gautam]

i am using taq pol  fermentas

0.2micro molar primer

50 ng template

[phage434]

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?

[Trof]

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

[ascacioc]

http://www.fermentas.../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

[Trof]

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.

[leelee]

Trof, on 04 September 2012 - 12:18 AM, said:

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.

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

[ascacioc]

@ 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

[phage434]

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.

[Trof]

@ascacioc: As far as I know, the 100 ng of human gDNA in reaction is like golden standard. When I'm working with other templates I recalculate to equal number of target gene copies, because It's actually number of copies that matter (however I understand it's more complicated than this, since generaly more complex templates amplify differently). Usually using my web aplication for copy number.

But practically gDNA is not that sensitive to the exact amount in reaction. I usually don't even measure DNA before PCR and just put there 1 ul, it works It will work with 50 ng, 300 ng, 500 ng.. generaly not more than 1 ug. Since all isolation procedures and blood volumes we use are similar, the concentration doesn't vary that much, usualy it's around those 300 ng/ul. In that case I don't bother to dilute it for PCR and specific concenetration, because it doesn't matter. If there is extremely high concentration then I dilute, because reaction would fail.

Of course there are methods where the concentration should be adjusted, for any real-time quantitation generaly less than 100 ng is used, because it's very sensitive. Especially with SYBR assays the increased overal DNA concentration causes high background level fluorescence. For special aplications like HRM even less is used, between 5-30ng in reaction, all samples adjusted to the same concentration.

When you ask someone how much gDNA to put in reaction, he will usually tell you 1 ul as a joke. It's not a joke though

[leelee]

ascacioc, on 04 September 2012 - 04:30 AM, said:

we never know what the people asking here are actually using as a template

Yeh, that's what I meant, that we don't know so it could equally be plasmid or genomic template- but I was also having a bit of a joke with Trof and never meant my post to sound too serious

Edited by leelee, 04 September 2012 - 06:46 AM.

[Janesh Gautam]

i am using At genomic DNA as a template and i have also tried with 3% dmso this time and annealing 58,60 62 64 66 68 and early denaturation 95 3 min and extension 72 for 1 min
my amplicon size is 1.5kb

[ascacioc]

At = arabidopsis thaliana? I know that the plant genome is a bit tricky because it is GC rich. Now, a lot of what you are stating makes sense. Indeed you need a modified protocol and still it will not work. I hope there is someone here with knowledge about it because in between the people who replied until now, I don't see smb with experience in plants genomics (I hope I don't offend anybody here, but Trof human genomic DNA as far as I understand, for me synthetic genes that come in little plasmids is the way...)

[Trof]

I have worked with human GC rich regions only, but 5% DMSO was the best fit for most, you can try up to 1 0%. It still amplified but didn't had such bright product. In my case 1 % was not enough, if you have more GC rich template, maybe 3 % is still not enough, try more concentrated.
What about the Primer3 Tm values? They usually work for me with Ta -4 degrees from this calculation. Tm's can vary a lot, that's why I'm asking about algorithm I'm comfortable with.

And another thing, did you checked the DNA is free of inhibitors? Do you have any working assay on the same species, so you can exclude potential problems with template?