Login

Join for Free!
118870 members


qPCR

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderator: BioTeam

qPCR

Postby xiaolong88 » Tue Dec 11, 2012 4:58 pm

Hi,

I have transcript (RNA) which I would use for qRT PCR for standard curve-the standard.
I should quantitate the transcript (RNA) before reverse transcribe them into cDNA.
I wonder I should serially dilute the transcript and perform reverse transcription? Or I should synthesis the cDNA first and only perform serial dilution for qPCR? Any different? :roll:

Hope you could help me :)
xiaolong88
Garter
Garter
 
Posts: 9
Joined: Mon Feb 23, 2009 5:20 pm

Postby JackBean » Tue Dec 11, 2012 11:00 pm

you should first make the cDNA as soon after RNA extraction as possible and then you can handle it as you want ;)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5689
Joined: Mon Sep 14, 2009 7:12 pm

Postby bravebeaker » Wed Dec 26, 2012 4:47 am

1. extracted RNA --> DNase treatment --> RT to synthesize cDNAs
2. use same primers to amplify your target by PCR
3. extract bands from gel and purify target DNA
4. use the purified DNA for making a standard curve.
5. runa a qPCR
AHH, Gravity! Such a cold cruel mistress.
User avatar
bravebeaker
Garter
Garter
 
Posts: 44
Joined: Sun May 08, 2011 3:26 am
Location: Kobe, Japan



Return to Molecular Biology

Who is online

Users browsing this forum: No registered users and 0 guests