Hi all,
I'm fresh in running PCR. I have an enquiry about the PCR cycling program on detection genus of Chlamydia.
I'm using the primers stated its Tm is 56-58C.
however, the PCR cycling conditions are as follows
1. 10min at 94C
2. 15sec at 95C for 40cycles
3. 15sec at 55C
4. 30sec at 72C
I have no product for this conditions at the end. I'm thinking to do a gradient to measure the optimum temp for annealing
Please advice for other options. Thank you very much.
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icarus.rachel's Content
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#61957 Chlamydia PCR cycling conditions
Posted by
on 09 March 2010 - 08:25 PM
in
PCR, RT-PCR and Real-Time PCR
#48829 multiplex PCR
Posted by
on 01 December 2009 - 07:54 PM
in
PCR, RT-PCR and Real-Time PCR
Hi all,
It is about running the multiplex PCR. When I did the PCR separately, the band can be run individually. However, when I mix two primers set together, and half the total vol.. I cannot see any bands in the gel. Could you please offer any suggestions so that to improve? Thank you.
Did your primers self-binding together? (Primer Dimer)
Tell us more of your protocol so that we can help you more...
I got the primers, 402bp (Tm: 66-77C), 308bp (Tm: 76C) & 245bp (Tm: 77C). with annealing 58C for 30s (according to protocol).
If I decrease the annealing temp, how low should i adjust?
if i decrease PCR primer & increase the template? but they work when do it in separate.
what about increase MgCl?
Thank you very much in advance.
#48631 multiplex PCR
Posted by
on 30 November 2009 - 11:55 PM
in
PCR, RT-PCR and Real-Time PCR
Hi all,
It is about running the multiplex PCR. When I did the PCR separately, the band can be run individually. However, when I mix two primers set together, and half the total vol.. I cannot see any bands in the gel. Could you please offer any suggestions so that to improve? Thank you.
It is about running the multiplex PCR. When I did the PCR separately, the band can be run individually. However, when I mix two primers set together, and half the total vol.. I cannot see any bands in the gel. Could you please offer any suggestions so that to improve? Thank you.
#36267 ELISA - calculate diluted concentration
Posted by
on 13 September 2009 - 05:31 PM
in
ELISA and Immunoassay
i use the Ln(Absorbance) against Ln(conc) and generate a equation in excel. It looks good when compared with the standard.
So could you give some advice on calculate the concentration? instead of 1:10000, I make 1:10 by mistake.
Thank you very very much.
So could you give some advice on calculate the concentration? instead of 1:10000, I make 1:10 by mistake.
Thank you very very much.
#36227 ELISA - calculate diluted concentration
Posted by
on 13 September 2009 - 07:39 AM
in
ELISA and Immunoassay
Hi, when the standard curve is already plotted, how to calculate the concentration from a 1:10 diluted samples? is it just multiplied by 10 as final concentration? but i cannot get the consistent result when i'm doing 1:2, 1:4, 1:8. Please suggest. Thank you.
