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Tomato Actin PCR+Images included


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#1 Fluffy

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Posted 29 July 2011 - 01:53 PM

Hello All

I did a tomato Actin PCR and the following were my primers:
Forward Primer 5'-TTC CCT CTA TGC CAG TGG AC-3

Reverse Primer 5-CAT CTC CAG AGT CCA GCA CA-3

The actin gene is Tom52 and accession is U60482 (to find use genbank)
I did not design the primers myself but I got them from an article and the article link is My link

The PCR product length is 271 nucleotides.

I have attached gel images of my PCRs. My question is I am not sure whether amplification of my desired PCR product happened? From what I see, it seems like there are bands, but are they the desired bands with the right size? The size when compared to DNA ladder makes me think its not my band of interest since size is not correct I believe. Please help me clarify this issue.

Gel pictures depicting same PCR with different DNA ladders:
Image 1: first lane is 10kb DNA ladder, only look at the next 8 lanes for my PCR. Don't worry about last 8. Based on picture, is this the right size bands (271)? Are they my PCR product or just background (primer dimers)? 7.19.2011 new actin gel.jpg

Image 2: first lane is 1kb plus DNA ladder, two bands in the middle are my PCR runs, last lane is 1kb DNA ladder. So? what do you guys think? Are they the proper bands with proper size? 07.21.2011 actin pcr 07.20.2011 rerun YA primers.jpg


I would greatly appreciate any help in this,
Thanks
Yasamino

#2 ivanbio

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Posted 29 July 2011 - 07:25 PM

Hi Yasamino,

If I remember my 1kb DNA ladder the smallest size marker is 100 bp. If you look at Image 2 your PCR "bands" are below the 100 bp size marker, so most likely these are not PCR products but primer dimers. The fact that your "bands" are much fuzzier than the bands that make up your size ladder also suggests that you are looking at primer dimers, not PCR products.

Ivan
Carlsbad, CA

#3 Fluffy

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Posted 02 August 2011 - 08:42 PM

Hi Yasamino,

If I remember my 1kb DNA ladder the smallest size marker is 100 bp. If you look at Image 2 your PCR "bands" are below the 100 bp size marker, so most likely these are not PCR products but primer dimers. The fact that your "bands" are much fuzzier than the bands that make up your size ladder also suggests that you are looking at primer dimers, not PCR products.



Hello Ivan

Thanks a lot for your response. I believe you are correct, I tried another set of actin primers and gladly I got my right size bands.
Thanks again,
Yasamino




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