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PCR product size confusion

PCR product size genes

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3 replies to this topic

#1 MarieJones2291

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Posted 10 February 2014 - 04:27 PM

Hi!

I know I'm a bit annoying as I asked similar question some time ago but it's all due to the lack of literature to research or at least I cannot find much.

Basically I performed PCR to amplify the tetracycline resistance gene from pBR322. I was given primers to use:

5’-CGCAGTCAGGCACCGTGTATG-3’
5’-CCATTCAGGTCGAGGTGGCCC-3’

The gene itself is 1,191 bp long. However, I used some of the softwares to predict the product size, using the entire plasmid sequence and given primers and the predicted product size was 1,200 bp or something similar. After having performed PCR and running the gel I did notice bands located between 1000 - 1500 bp according to the DNA ladder I used. 

I am not really sure if I predicted the correct size of the PCR product and whether the fact that the product is slightly bigger than gene itself means I amplified the wrong thing. Sorry for being repetitive but this is really confusing to me.

Thanks!



#2 IstiakM

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Posted 10 February 2014 - 06:22 PM

Hi,

You can take your PCR product and subject it to a sanger sequencing, and after that BLAST it with nucleotide database to see, whats the actual sequence is? In this way you can be sure. I had once face such confusion when I was trying to amplify a human exon of 255bp, but my gel showed a 155bp amplification. When ran it in a Sanger Sequencing and BLAST it with nucleotide database, I found that the sequence is 98% identical to a bacterial sequence. I am sending the link of my paper on this and you can have some details on that matter.

 

http://www.biomedcen...1756-0500/6/481

 

Kind Regards

Istiak



#3 bob1

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Posted 11 February 2014 - 12:31 AM

The predicted size  is not necessarily the size it will run on the gel, as there are a few different thing that can affect migration, salt being one of the more common.  There won't be a visible difference between 1,191 and 1,200, no matter what % gel you run.

 

As IstiakM says - sequence it to confirm, or do a restriction digest analysis (if you can find useful sites that give meaningful size products).



#4 MarieJones2291

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Posted 11 February 2014 - 05:06 AM

Thank you. Now it's quite clear to me. This was my first PCR ever so I had no experience I could use to detect any abnormalities but your advice been helpful. Thanks!







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