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-   -   PCR troubleshooting (http://www.molecularstation.com/forum/pcr-polymerase-chain-reaction-forum/84449-pcr-troubleshooting.html)

dlise 01-13-2012 06:25 AM

PCR troubleshooting
 
1 Attachment(s)
Hi there!

I've been working on this gene (wolbachia gene) for nearly half a year now and over these months, I've been having problems with my wsp primers. some days it'll be as good as anything, and some sucks.

We have tried changing the polymerase from Taq pol to Phusion high fidelity polymerase. it was working for a while and now it's gone chaos again.

I have attached a photo of my latest screening. the last two lanes are positive control and negative control consecutively.

I was wondering if anyone could give me an idea as to why there're more than one bands and why are they so many bands at the bottom. I shouldn't be seeing any other bands after ~560bp (expected band size).

FYI, DNA were extracted only 2 days prior to this screening, all reagents have been freshly made for troubleshooting.

thanks in advance!

butters 01-14-2012 03:49 PM

Re: PCR troubleshooting
 
Can you attached a gel picture where the result is as good as anything?

Form first glance it seem like your PCR or your primer design is/are not optimized. Did you change anything like PCR machine or carry your PCR mix preparation as fast as possible (leaving your mix too long after adding DNA template+Taq may result in non specific amplification)?

dlise 01-15-2012 10:53 PM

Re: PCR troubleshooting
 
1 Attachment(s)
Hi butters,

I did everything the same since i first started working with PCR - doing everything on ice, and make sure everything's in the pcr machine as quickly as possible.

The primers I used are actually a surface protein. so, it may detect more than one wolbachia gene. however, their sizes would be between 500-600bp.

so far, the attached is the best Ive ever gotten with this primer

thanks again!

admin 01-16-2012 12:29 AM

Re: PCR troubleshooting
 
http://www.molecularstation.com/foru...husion-mix-jpg
Hi
IF you aren't getting your product then you have either a problem with your primers or your PCR amplification or your DNA template.

Have you tried to optimize your PCR? what are all the lanes labeled?

dlise 01-16-2012 03:54 AM

Re: PCR troubleshooting
 
Real sorry for not labelling the image!

Lane 1-5: Cx. annulirostris mosquito samples
Lane 7-11: Ae. albopictus mosquito samples
Lane 13-17: Ae. notoscriptus mosquito samples
Bottom lane 1-5: Culicoides brevitarsis midge samples
Bottom lane 7: positive control
Bottom lane 8: negative control (possible smearing?)

I am getting my product, but the problem is that most of my products are accompanied with a trail of smearing or even with smearing in negative controls.

admin 01-16-2012 08:08 AM

Re: PCR troubleshooting
 
It makes more sense now. Seems you may have both some contamination (or mixing of the loading into the negative control its possible) and primer dimers which are competing with your PCR main reaction.

I would tighten up your PCR by optimizing either increasing/changing your annealing temperature / PCR conditions and also trying fresh buffer/template so you don't waste more time with possibly contaminated reagents

best wishes

dlise 01-16-2012 11:19 PM

Re: PCR troubleshooting
 
thanks for that. i'll give it a go and see what happens.

davidflora 12-20-2012 08:02 AM

Re: PCR troubleshooting
 
[Only registered and activated users can see links. Click Here To Register...]to generate large amounts of a desired product can be a double-edged sword. Failure to amplify under optimum conditions can lead to the generation of multiple undefined and unwanted products, even to the exclusion of the desired product. At the other extreme, no product may be produced.


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