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Primer Design, help im New


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#1 Margot Schikora

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Posted 03 June 2013 - 08:15 AM

Hello there, i need your help, i´m new in this forum and new to PCR.

I´m going to tell you my Isuue, you probably going to smile because the solution is very easy :-D

I´m going to do qRt-PCR for GFP; i have a pAc-GFP1-N1 plasmid Vector wich i`m introducing into cells. After an inkubationtime and isolation of total RNA i´m going to do qRT-PCR.
So my problem, in order to do amplification of the AcGFP mRNA, which is translated with an oligT-Primer into c-DNA, i need Primers for the c-DNA.

But i can´´t find the sequence for the AcGFP1-gene of the cDNA. I studied chemistry so my biology knowlege is, not very good:-D

i found programs that calculate the best Primers for ou but i Only have the Sequence of the gene on the plasmid.

What should i do know, because writing down the whol 500 basepairs manually can´t be the right thing to do.


Pleas Help!!

Thank you

#2 phage434

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Posted 03 June 2013 - 09:15 AM

If you have sequence for the gene being expressed, that is enough. You don't need the sequence for the entire plasmid. Take your gene sequence and put it into the primer3 program and let it choose primers for you, making a 300 bp or so product. Those primers should be fine. http://frodo.wi.mit.edu/

#3 Margot Schikora

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Posted 03 June 2013 - 01:21 PM

hey thanks for your help, but aren´t these the wrong primers, because the gene is translatet to mRNA which has the same sequence than the Gene and this ist made to a complementery cDNA during the PCR experiment. So i need primers for the complementery strand of the gene Sequence. Or am i wrong in this case?
and i can´t find the sequence of the cDNA or a Program which can konvert the Genesequence into it.

Thank you so much

#4 phage434

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Posted 03 June 2013 - 02:28 PM

The sequence of the cDNA and your gene and the mRNA are all the same (modulo T<->U issues, and reverse complement issues). Primers will amplify either strand of the DNA (one primer binds to each strand, and makes the complementary strand, which then has sites available for the other primer to bind to).

#5 Margot Schikora

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Posted 04 June 2013 - 01:05 AM

ahhh yeas sure...now i see..it doesn´t matter i should have checked the primer sequence...the reversed prime ris for the complementery strand and then there will be amplifid a new strand for the forward primer....oh thanks a lot, i was thinking the wrong way! thank you!!




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