PCR vs RT-PCR

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

Moderators: Leonid, amiradm, BioTeam

Post Reply
magicsiew
Coral
Coral
Posts: 145
Joined: Sun Jan 24, 2010 3:07 pm

PCR vs RT-PCR

Post by magicsiew » Sun Oct 03, 2010 1:12 pm

Hi, sometime in experiment, when carrying out PCR, no band at gel electrophoresis, but when carrying out RT-PCR, band visible at gel electrophoresis. What I don't understand here is, in PCR, the DNA I used is genomic DNA, while in RT-PCR, RNA was used, which become cDNA after reverse transcript. In other word, RT-PCR cDNA sample is shorter than gDNA in PCR, with less location(specific or non-specific) for the primers to complementary bind to. My question will be why the gene get to amplified in RT-PCR, but not in PCR?

User avatar
JackBean
Inland Taipan
Inland Taipan
Posts: 5694
Joined: Mon Sep 14, 2009 7:12 pm

Post by JackBean » Sun Oct 03, 2010 3:20 pm

well, the gDNA is fairly long piece of DNA, so
1) it can brake, if not handled carefully
2) it may not be amplified, if the introns are long and amplification time is not long enough
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

User avatar
JackBean
Inland Taipan
Inland Taipan
Posts: 5694
Joined: Mon Sep 14, 2009 7:12 pm

Post by JackBean » Sun Oct 03, 2010 3:20 pm

also, get propriate amount of DNA into the tube is a little harder. Did you try to cut it a little with some RE?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

magicsiew
Coral
Coral
Posts: 145
Joined: Sun Jan 24, 2010 3:07 pm

Post by magicsiew » Sun Oct 03, 2010 4:32 pm

I din't cut it, but I dilute it to 100ng/ul.

In my other case, the degenerate primers able to amplify the gene in PCR, I got 3 set of primers here, each with 900bp, 3500bp and unknown product length respectively. The bands form at AGE show ~800bp, ~2500bp and ~2500bp.

For the 1st 2 sets, is it likely that the product could be my gene of interest? Or just some non-specific binding? I will do cloning and send out for sequencing later, but before this, I am not really sure for the 2nd 1, is about 1000bp difference.

For the 3rd set, any idea to determine if this amplification is the correct 1? I don't know the product length because the forward primer and reverse primer are from different alignment.

User avatar
JackBean
Inland Taipan
Inland Taipan
Posts: 5694
Joined: Mon Sep 14, 2009 7:12 pm

Post by JackBean » Mon Oct 04, 2010 10:58 am

you may try to digest it partially, that should increase the availability
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

magicsiew
Coral
Coral
Posts: 145
Joined: Sun Jan 24, 2010 3:07 pm

Post by magicsiew » Thu Oct 07, 2010 2:04 pm

Hi, now I have 7 sets of primers, set 2,3, and 5 show band in PCR, but in RT-PCR, set 5 and 6 show band only. No problem with positive and negative control. Now I am confused in which one should I send for sequencing? Of course will be better if can send all of them, but running low budget.

User avatar
JackBean
Inland Taipan
Inland Taipan
Posts: 5694
Joined: Mon Sep 14, 2009 7:12 pm

Post by JackBean » Mon Oct 11, 2010 9:17 am

do some restriction analysis ;) also, by combining these fragments, you should get one large piece, if are they homologous ;)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

Post Reply

Who is online

Users browsing this forum: No registered users and 4 guests