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PCR with mammalian cells as template

PCR mammalian cell

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#1 Hellen

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Posted 02 March 2015 - 06:52 PM

Hello everyone, I need your help on my work recently. A constructed plasmid was transformed into Hela cells. The vector is used to expressing protein. Between promoter and GFP, there is a transposon-like sequence with TAA. So if the transposon sequence is excised, GFP can be expressed. Now I want to confirm the transposon excision with PCR and sequencing. I used Hela cells transformed by the plasmid as PCR template, and primers are designed according to the vector. I thought to achieve the PCR product that is shorter than original construct. Then the product can be recycled and used to sequencing. However, I obtained nothing. Why? I am confused. Someone told me that I should achieve the RNA, then RT-PCR and PCR with cDNA as template.I have little experience on these. Could you give me some useful information or your critical experience about these? Thank you very much.

#2 bob1

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Posted 04 March 2015 - 01:31 PM

So you are looking to check that the excised product is no longer in the plasmid? If you did this, then I would assume that the plasmid is no longer circular, and as such won't give you a product if you are trying to amplify across the vector ends.

 

If you are thinking that the transposon is excised after transcription, then you will definitely have to extract RNA, make cDNA and the PCR.

 

However, assuming that the mechanism is correct, that GFP can only be expressed when the transposon is excised, then surely GFP expression is all you need to see (though RNA would confirm it).







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