facing problem with pcr gel results

[jizzs]

i have been running pcr gel for a 1.3kb gene but getting nothing except long smears with no primer dimers at end .

i dnt know what is the problem ......

[Cathal Garvey]

I'm afraid that's hard to diagnose at the best of times, let alone
without some critical info:

- Can you verify the integrity/identity of your template DNA? I spent
ages trying to get a PCR to work for a gene once, only to come to the
conclusion that the DNA wasn't what I was told it was to begin with. A
synthesised copy amplified fine.
- What enzyme did you use? Taq is rubbish at long extensions, and may
fail to correctly amplify 1.3kb
- Streaking can be a sign of nuclease digestion; did you prepare, run
and gel analyse the DNA in short order, or was your DNA/reaction left
for any length of time between start and finish?
- What kind of water did you use for your PCR? Tapwater is
inappropriate, but deionised (not distilled) water or better should be fine.
- Have you or anybody else ever successfully amplified with the primers
you're using? It could be a primer issue!

I'd ask questions about your gel if the ladder were as smudged as the
PCR results. Looks like your issue is either the source DNA, or the
reaction.

Sorry for your troubles!

On 15/05/12 11:53, jizzs wrote:
> i have been running pcr gel for a 1.3kb gene but getting nothing except
> long smears with no primer dimers at end .
>

> <https://lh4.googleusercontent.com/-NMAmf4dE7JQ/T7I1sZqosVI/AAAAAAAAAAY/cAWDcW58otY/s1600/PCR+FSZB+14.5.12.tif>

> i dnt know what is the problem ......
>

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[jizzs]

ya am using taq, and deionised water, and about g dna this is the pic of same sample.....

[shamrock]

Is this your template DNA? Genomic? Same amount that you are using in the PCR? If so, I would suggest dialing the template concentration waaay back. Try a titration with several 10 fold dilutions of the template and see if the results improve. 

Looks like there are some faint bands among the smear - do you have the ability to do a temp. gradient to look at different annealing temps? If not you could try various Mg concentrations or DMSO (I've had good luck with DMSO in high GC templates). Also if your adding everything (template, primers, enzyme) together you might be getting some nonspecific amplification early on. Go through a denaturation step first and then add the enzyme (Hot start). 

I've previously used Taq to amplify a 2.5 Kb fragment without any problems. 

Good Luck! 

[Cathal Garvey]

Cool! Did you do anything special to achieve this? I was of the
understanding that Taq tended to create mismatches and dissociate,
leaving loose 3' ends that can't anneal and continue polymerising. The
result is usually a failure to amplify larger templates. Figures I've
heard for amplification ceiling range wildly between 700bp and 1.5kb.

On 15/05/12 16:38, shamrock wrote:
> I've previously used Taq to amplify a 2.5 Kb fragment without any problems.

[shamrock]

OOp's looks like I'm going to have to take back that statement about amplifying a 2.5 Kb fragment with Taq. Checked my notebook and it turns out I had used Pfu and not Taq. 

[Nathan McCorkle]

can you label the gel lanes, other than the MW ladder I'm not sure what to make of anything

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[Jeswin]

I don't know where your gDNA is coming from, but maybe you got some DNAse in it? What is this sample from? How was it collected/stored?

[jizzs]

its dictyostellium genome... finally resuspended in 1:5 TE buffer.

On Wednesday, May 16, 2012 4:53:11 AM UTC+5:30, phillyj wrote:

I don't know where your gDNA is coming from, but maybe you got some DNAse in it? What is this sample from? How was it collected/stored?

On Tue, May 15, 2012 at 1:47 PM, Nathan McCorkle <nmz...@gmail.com> wrote:

can you label the gel lanes, other than the MW ladder I'm not sure what to make of anything

On Tue, May 15, 2012 at 6:53 AM, jizzs <jitesh...@gmail.com> wrote:

i have been running pcr gel for a 1.3kb gene but getting nothing except long smears with no primer dimers at end .

i dnt know what is the problem ......

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Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

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[John Patton]

Taq should be able to amplify that just fine, I do it regularly in the lab.

Couple of questions:

Concentration of genomic DNA, Taq can be temperamental with too much template?

PCR protocol, temperatures times etc?

Tm of Primers?

Salt recipe?

John

[Zebedee Boy]

What's the difference between the lanes with primer-dimer and the lanes with the broad range smears? To me this looks like your primers aren't working as they should.
 
I'd suggest,
 
1) if you haven't already, blasting your primers to ensure that they are a monospecific in the Dictos genome and, if the resource is out there, ensure that they aren't on top of any known SNP or CNV.
2) Include a different working PCR reaction and an NTC so you can exclude the PCR reagents/reactions as the problem.
3) Look at your reaction conditions if you have a gradient cycler start with a temp curve and Mg profile.
4) As Cathal suggested, try a different polymerase
 
Zeb

---

Date: Wed, 16 May 2012 23:21:02 -0700
From: jitesh...@gmail.com
To: diy...@googlegroups.com
Subject: Re: [DIYbio] facing problem with pcr gel results

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