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PCR, using gel extracted band as a template


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#1 wincher

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Posted 30 January 2011 - 02:56 PM

Hello

I'm running a PCR reaction to amplify a receptor cDNA from pcDNA3.1 plasmid. Unfortunately, beside the correct band I get also unspecific products. I guess it's due to not sufficient primer specificity and I will order a new primers soon.

But anyway I considered isolating the band of interest from the gel and repeating the PCR using expected product as a template. I would like to know your opinion on that idea. Is it worth of trying? What should be the amount of template used in this case (the product is 1,2kb size) and if the annealing temperature will be affected somehow?

Thank you in advance for your responses.
Regards
TK

#2 Adrian K

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Posted 30 January 2011 - 07:00 PM

Hi there,
I don't see any reason on why you can't do that... But then you must have "just enough" DNA: not too much and not too little DNA to begin with.
Just make sure to sequence your product to verify any DNA mispairing as it very prone to happen.

If you want to do cloning, I think after you had gel excise the product, you can directly clone it into a vector, no need to do again your PCR.
Adrian
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..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

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#3 leelee

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Posted 30 January 2011 - 09:11 PM

I've done that before. Should be fine.
Might be worth optimising the original PCR though. Have you tried increasing your annealing temp to see if that gets rid of the non-specific binding?

#4 wincher

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Posted 31 January 2011 - 04:38 AM

Hello again.

First of all thank you for your responses.

@adrian: "not too much and not too little DNA" ;) do you have any suggestion which amount to start with? more or less than 50 ng?
Concerning the next step, I mean ligation - I assume that I will have to repeat it several times (vector and insert are of big size and it will probably not work at the first time) so it would be too much time consuming to isolate PCR product from gel every time, that's why I'd like to get the insert directly from second stage PCR.

@leelee: PCR have already been optimized with relation to annealing temperature, Mg2+ and template concentration.

tk

#5 Adrian K

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Posted 31 January 2011 - 04:56 AM

Hi wincher,
I have to apologize for not being clear. Actually I did not quantify my template most of the time, my measurement is by look into the gel picture. As long as you not see a too thick band and not a too faint band, it is sufficient.

I suggest you to clone into TA or blunt end cloning vector (I mostly use CloneJET™ PCR Cloning Kit, Fermentas), sequence it to check the nucleotides, and use the vector as template and do PCR on it. This is how I deal with some difficult template. Else you can do as what you had planned.

All the best.

Adrian
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434




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