Login

Join for Free!
118488 members


RT-PCR

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

Moderator: BioTeam

RT-PCR

Postby banuy » Mon Jan 03, 2005 4:42 pm

I am using SuperScript First-Strand Synthesis System for RT-PCR. I performed the reaction with control RNA and it worked. I tried 4 different primer sets for 4 different genes. 3 of the primer sets which amplify smaller than 500 bp fragments are working but 1 primer set which should amplify a 2 kb fragment is not working. Are there anyone who faced with problems with moderately long product amplifications?
banuy
Garter
Garter
 
Posts: 1
Joined: Mon Jan 03, 2005 4:40 pm

Postby cathbr » Mon Mar 12, 2012 3:34 pm

Hi,

a lot of things could happen between the RNA and your PCR... If you give me more details I might be able to help
For ex:
the conditions that you are using for the PCR
if you are using specific or random primers for the RT
if you try to amplify the same products, or spliced varients,... with 4 different primers...
Are you sure that your 2kb fragments is within the cDNA that you created?
Did you try to increase the elongation time?
Did you try different annealing temperatures?
cathbr
Garter
Garter
 
Posts: 7
Joined: Mon Mar 12, 2012 1:38 pm

Postby bravebeaker » Wed Jan 23, 2013 8:24 am

as far as i know and correct me if i am wrong, RT-PCR amplification products should be short, not too short but not too long, if I understood correctly you are amplifying a 2Kb amplicon I think that is very very long.
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


Re: RT-PCR

Postby JackBean » Wed Jan 23, 2013 3:27 pm

why should it be short?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5678
Joined: Mon Sep 14, 2009 7:12 pm

Postby bravebeaker » Mon Jan 28, 2013 10:18 am

I am saying based on the kit I am using which is SYBR green by TOYOBO and also many qPCR protocols over the internet as well as papers indicate the use of a short amplicon between 80 to 200 bp... what do you think? maybe banuy's experiment is different?
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

Postby JackBean » Mon Jan 28, 2013 1:49 pm

First strand synthesis and qPCR are IMHO something different. Yes, for qPCR one should have only short amplicons, but I don't see reason why should you amplify only short pieces during reverse transcription.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5678
Joined: Mon Sep 14, 2009 7:12 pm

Postby bravebeaker » Tue Jan 29, 2013 1:19 am

Now I see your point, but in my case I use the primer mix that comes with the RT kit by Takara..
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

Postby JackBean » Tue Jan 29, 2013 11:39 am

random hexamers?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5678
Joined: Mon Sep 14, 2009 7:12 pm

Postby bravebeaker » Wed Jan 30, 2013 4:27 am

yes
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

Postby JackBean » Fri Feb 01, 2013 11:18 am

in such case you will have mixture of random lengths and only the length of amplification step will influence the maximal length of your amplicons
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
User avatar
JackBean
Inland Taipan
Inland Taipan
 
Posts: 5678
Joined: Mon Sep 14, 2009 7:12 pm


Return to Molecular Biology

Who is online

Users browsing this forum: No registered users and 1 guest