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The slightly bigger band dillema.

PCR electrophoresis gel molecular marker

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#1 Tick

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Posted 16 January 2015 - 12:10 PM

Dear All,

 

This is my first post on this forum and I'm looking forward to discussing all sorts of my, and other problems, findings etc!

I've done an RT-PCR on the PML-RARA translocation in the human NB4 APL cell line. I blasted my primers and the expected fragment length should have been 128 bp. I loaded my samples on an 1% agarose gel, along with Molecular weight marker 8 (from Roche I believe. The molecular weight marker is distorted, I accidently put the tip of the pippette into the gel itself when loading the samples.

The problem is, my amplification product, isn't quite 128 bp, but based on the mm8, it is more or less around the 140 bp (quite the difference). I can't really explain why this is, apart from my poor pippeting skills.

 

I enclosed the pic of the electrophoresis for your viewing pleasure!

 

Thanks in advance for answers and solutions, and for your interest in this topic!

 

-Tick-

 

PS. The RNA was isolated using Tripure reagent and cDNA was made using the cDNA first strand synthesis kit by Roche!

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#2 CPRES

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Posted 16 January 2015 - 01:37 PM

I don't know why, but to me it looks like exactly where 128 bp should land. Seriously.

 

Running such short fragments on 1% agarose gel to unequivocally validate a fragment size is not realistic. You may need to run a higher % agarose gel or a polyacrylamide gel. The best way to confirm the valid product is not size alone but the sequence, so you should get it sequenced from both ends which will also tell you the size.

 

You are doing a good job, keep it up!


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#3 Trof

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Posted 17 January 2015 - 12:22 PM

My opinion is you can hardly try to tell the difference of 128 and 140 bp from this picture, the marker bands are too close, but in translocation I would always sequence the product, cause the breakpoint may differ actually. 

Also salt content has an effect on the band mobility, and the marker surely don't have identical salt content as your sample. In case of RFLP analysis this is very obvious (restriction buffers are high salt). Running the gel longer difuses the salts, so for quality size measurement you should have marker over wider range of gel, but probably not on 1% agarose, the picture looks pretty limiting for your sizes.

 

For small PCR/RE products in general, you may use 2% agarose gel with SB buffer that can run with higher voltage. I have been runing RE fragments below 100bp with it very successfully, sharp and bright.


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