Search found 9 matches

by frankalpha
Thu Sep 23, 2010 2:33 pm
Forum: Molecular Biology
Topic: The right temperature for unfolding RNA secondary structure
Replies: 3
Views: 2458

Re: The right temperature for unfolding RNA secondary structure

Sure. Say if I don't put the enzyme in at first, just heat the RNA to unfold the secondary structure, what temperature should I use to best unfold the secondary structure without damaging the RNA? And if the temperature drops down (I will have to add enzyme anyway), will the structure re-form?
by frankalpha
Wed Sep 22, 2010 9:10 pm
Forum: Molecular Biology
Topic: The right temperature for unfolding RNA secondary structure
Replies: 3
Views: 2458

The right temperature for unfolding RNA secondary structure

As the title goes, what's the right temp. for that? In RT using superscript II or III, we use 65°C. Can we go any higher and will this damage the RNA? Thanks.
by frankalpha
Wed Sep 22, 2010 9:05 pm
Forum: Molecular Biology
Topic: Reverse transcription of a circular RNA template
Replies: 7
Views: 6849

Re: Reverse transcription of a circular RNA template

Actually, I want to get ds cDNA from RNA. I've figured it out. There are several kits available that can synthesize ds cDNA from RNA (there is a step for second-strand cDNA synthesis after the first-strand synthesis). Thank you for your help.
by frankalpha
Tue Sep 21, 2010 1:57 pm
Forum: Molecular Biology
Topic: Reverse transcription of a circular RNA template
Replies: 7
Views: 6849

Re:

And where will you get the hairpin? You're not talking about RT(reverse transcription)-PCR? No, I'm not talking about RT-PCR, just RT instead. The hairpin theory came from this link 'http://dwb4.unl.edu/Chem/CHEM869N/CHEM869NLinks/www.dur.ac.uk/~dbl0www/Staff/Croy/cDNAfigs.htm'. The 2nd paragraph a...
by frankalpha
Tue Sep 21, 2010 1:54 pm
Forum: Molecular Biology
Topic: How to get double stranded cDNA from first-strand cDNA
Replies: 5
Views: 4384

Re:

JackBean wrote:well, any polymerase would be fine having some primer


How's the reaction condition? Just add the polymerase and use the assumed primer and set probably 20 PCR cycles, i.e. denaturation - annealing - extension?
by frankalpha
Tue Sep 21, 2010 1:52 pm
Forum: Molecular Biology
Topic: How to get double stranded cDNA from first-strand cDNA
Replies: 5
Views: 4384

Re: How to get double stranded cDNA from first-strand cDNA

BeyazRus wrote:How long your cDNA strand is? Answer changes according to length.


The strands are 10 kb and 6 kb respectively. Thanks.
by frankalpha
Mon Sep 20, 2010 8:08 pm
Forum: Molecular Biology
Topic: How to get double stranded cDNA from first-strand cDNA
Replies: 5
Views: 4384

How to get double stranded cDNA from first-strand cDNA

As the title goes, if I want to get double stranded cDNA from the single stranded first-strand cDNA without conducting a PCR, What should I do? According to this link 'http://dwb4.unl.edu/Chem/CHEM869N/CHEM869NLinks/www.dur.ac.uk/~dbl0www/Staff/Croy/cDNAfigs.htm', seems I can use DNA Polymerase I to...
by frankalpha
Mon Sep 20, 2010 5:52 pm
Forum: Molecular Biology
Topic: Reverse transcription of a circular RNA template
Replies: 7
Views: 6849

Re: Reverse transcription of a circular RNA template

Thank you for your reply. I use only one primer for the reverse transcription and there is no ligase added, so the cDNA should be single-stranded and linear, right? If I want to get dsDNA, can I use DNA Polymerase I to extend the other DNA chain, using the hairpin loop at the end of the first-strand...
by frankalpha
Mon Sep 20, 2010 3:41 pm
Forum: Molecular Biology
Topic: Reverse transcription of a circular RNA template
Replies: 7
Views: 6849

Reverse transcription of a circular RNA template

As above, will the cDNA product be linear or circular (say if I use Superscript II or III)?
I suppose the cDNA product to be single-stranded. Am I right?