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icarus.rachel's Content

There have been 5 items by icarus.rachel (Search limited from 14-November 18)


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#48631 multiplex PCR

Posted by icarus.rachel 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. :P



#48829 multiplex PCR

Posted by icarus.rachel 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. B)


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.



#36267 ELISA - calculate diluted concentration

Posted by icarus.rachel 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. :)



#36227 ELISA - calculate diluted concentration

Posted by icarus.rachel 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.



#61957 Chlamydia PCR cycling conditions

Posted by icarus.rachel on 09 March 2010 - 08:25 PM in PCR, RT-PCR and Real-Time PCR

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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