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RT-PCR


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9 replies to this topic

#1 seed

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Posted 06 April 2011 - 02:53 AM

I am very confused with my results now. I have extracted RNA from tomato, synthesized cDNA and did PCR reaction. I also ran the PCR using gDNA. I got the same size of bands for my cDNA and gDNA. Supposely, my PCR product should be around 300bp and and my gDNA (with inton) is around 500bp. All the bands that I got are around 500bp. Than I repeated again my experiment because I thought that my samples have been contaminated with gDNA and I have tried varities concentration of MgCl2 for my PCR reaction (from 1mM MgCl2 to 50mM MgCl2. After ran the gel electrophoresis, I found that MgCl2 at concentrations of 25mM to 40mM showed that bands only for my cDNA, not my DNA and not my ubiquitin (housekeeping gene). Meanwhile, at concentration of 2.5mM, I got the band for my cDNA, ubiquitin from my cDNA and DNA, but not my gDNA sample. I also have ran together my control which are:
1) gDNA + cDNA
2) using random template to synthesize cDNA
3) PCR reaction without sample, only primer and PCR kit
4) PCR reaction using RNA
All the controls showed no bands.
Can someone help me with this? I have designed the primer using coding sequence (without intron)

#2 BioMiha

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Posted 06 April 2011 - 08:08 AM

what gel concentration did you use when separating the products? if the gel percentage is too low you might not see the difference

Edited by BioMiha, 06 April 2011 - 08:09 AM.


#3 seed

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Posted 06 April 2011 - 11:30 PM

i used 1%

#4 BioMiha

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Posted 07 April 2011 - 04:26 AM

do you have a Mw ladder to compare? I mean can you tell the difference in length?

#5 seed

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Posted 07 April 2011 - 05:25 AM

This is the picture of my gel for my RNA extraction after DNase removal. What are they, the one present on the well? I used hyperladder 1 as a marker.

Attached Thumbnails

  • RNA aftr treatment 060411.jpg


#6 seed

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Posted 07 April 2011 - 05:27 AM

This is the before the treatment

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  • RNA 050411.jpg


#7 seed

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Posted 07 April 2011 - 05:35 AM

Can you tell me whether my samples are contaminated with gDNA or not (based on the pictures that I have attached before)

#8 BioMiha

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Posted 07 April 2011 - 10:11 PM

Based on your pictures the bands you are getting are around 900 bp and 1400 bp. Also why do you have the same bands in both lanes? It seems likely that you are amplifying something non-specifically. In my experience when the primers for RT PCR are specific you should get only one band, however you can never eliminate all of your gDNA, therefore if the primers are not 100% specific you can indeed get some weird amplicons.

#9 seed

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Posted 07 April 2011 - 11:31 PM

sorry I didn't explain to you. The picture that I showed to you is just from my RNA extraction, before and after DNase removal. Not my PCR products. Sorry about that

#10 BioMiha

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Posted 08 April 2011 - 03:56 AM

Ok then I am probably just seeing the 28S and 18 rRNA. You can see in the pretreated samples the gDNA probably as the bands around 10000 bp which are gone in the treated samples. That means that most of the gDNA is gone. However some gDNA will ALWAYS be carried over because no elimination step is 100%. That's why intron spanning primers are used for RT PCR.




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