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Whole genome amplification from cDNA?


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#1 sweetstef

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Posted 04 July 2013 - 04:03 AM

My lab has been trying to amplify cDNA from single cells, through PCR. We've been having assorted ongoing problems, and now they are coming to me with a new protocol, but I'm not sure if this makes any sense. I am a new tech, and no one in my lab has any background in molecular biology, so any help is appreciated.

They want to take the RNA from the single cell, reverse transcribe it into cDNA, then use Qiagen kit REPLI-g, for whole genome amplification of single cells. Does this make sense to anyone? Are there any pitfalls that I should be aware of?

#2 pito

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Posted 04 July 2013 - 04:30 AM

My lab has been trying to amplify cDNA from single cells, through PCR. We've been having assorted ongoing problems, and now they are coming to me with a new protocol, but I'm not sure if this makes any sense. I am a new tech, and no one in my lab has any background in molecular biology, so any help is appreciated.

They want to take the RNA from the single cell, reverse transcribe it into cDNA, then use Qiagen kit REPLI-g, for whole genome amplification of single cells. Does this make sense to anyone? Are there any pitfalls that I should be aware of?

If I understand you correct you want to sequence the genome? Or ?

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#3 sweetstef

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Posted 04 July 2013 - 04:43 AM

Sorry if I wasn't specific. Our end result is not sequencing, we are really just looking to see if certain genes are expressed. We aren't doing qPCR because we don't care about expression levels, just whether or not certain genes are there.

#4 pito

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Posted 04 July 2013 - 06:29 AM

Sorry if I wasn't specific. Our end result is not sequencing, we are really just looking to see if certain genes are expressed. We aren't doing qPCR because we don't care about expression levels, just whether or not certain genes are there.

Ah ok, I understand it why they do it. By working with mRNA they know its expressed (at least at rna level). So yes its a common technique to use.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#5 sweetstef

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Posted 04 July 2013 - 10:41 AM

Great, thank you very much.




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