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Rt pcr

RT control Pcr

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4 replies to this topic

#1 shubhitamathur

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Posted 09 July 2015 - 10:09 AM

I have isolated RNA from HEK cell lines.While doing one step RT PCR using kit, and then running the gels, the negative control also shows a band at the same size as of desired for the sample.Why am I getting a band in negative controls?



#2 Mad Researcher

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Posted 12 July 2015 - 08:14 AM

Most likely due to contamination in one of your reaction component.

 

What about your positive controls?


Cheers,

Mad Researcher

#3 shubhitamathur

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Posted 14 July 2015 - 06:09 AM

Positive control also shows the respective band.The main problem is that why the band is observed in negative controls.I took all the precautions to set the reaction.There is a separate RNA working table.Still,there is a band in negative controls??



#4 almost a doctor

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Posted 14 July 2015 - 07:41 AM

What is your negative control?   Could it be showing DNA contamination in your prep? 



#5 shubhitamathur

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Posted 16 July 2015 - 09:06 AM

Negative control is cells only.....i.e cells in which no transfection took place.When there is no transcription,how can the desired band of 300 bp shows up there?







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