PCR vs RT-PCR

[magicsiew]

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?

[JackBean]

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

[JackBean]

also, get propriate amount of DNA into the tube is a little harder. Did you try to cut it a little with some RE?

[magicsiew]

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.

[JackBean]

you may try to digest it partially, that should increase the availability

[magicsiew]

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.

[JackBean]

do some restriction analysis also, by combining these fragments, you should get one large piece, if are they homologous