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joy123

Member Since 21 Oct 2011
Offline Last Active May 02 2013 09:48 AM

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Topics I've Started

urgent: need help with qPCR statistics

26 April 2013 - 08:06 AM

Hi, I did qPCR on 16S gene to test the level of a bacteria from four groups of people.

I don't have standard curve when I did qPCR. But in each plate I included one standard bacteria strain gene (in triplicate). I want to ask how should I analyze the data. Should I simply compare Ct value by nonparametric analysis? Or shoud I do some others (like delta CT?)?

I see one paper compare gene difference in patients and healthy control by "fold change". But I don't know what they used to compare. Just compare Ct?

I need the results be analyzed by the end of the day. Would you please help me with that?

Thanks a lotu

acceptable melt curve?

12 February 2013 - 07:36 AM

Hi I am doing qPCR. I wonder if you could help me judge if these melt cuve are good enough? I don't understand why there is a shoulder at ~74C and 70C. Is this set of primer specific enough for qPCR?

Thanks a lot!

good 16S univ. primer for qPCR?

29 January 2013 - 11:10 AM

Hi! Would you please suggest some good 16S univerisal primers for qPCR? I have difficult finding one and need it very soon.
I have tried a 466bp one, and it maybe too big.

Thanks a lot!

add template first or Syber mix first to reduce contamination in qPCR?

27 January 2013 - 09:02 PM

Hi, I have long been confused with contamination problem during PCR.

Now I plan to do qPCR. I want to ask for the 96-well plates, should I add templates in each well, and then add syber mix? Or if I should add syber mix first, and then add template in each well?

Thanks for your suggestions!

isoelctric gels hard to stain?

22 January 2013 - 07:31 AM

Hi, I have been experiencing problems staining a commercial isoelectric focusing gel.

I followed the staining method recommended by company: fix in 12% trichloroacetic acid+3.5% sulfosalicylic acid for 30min, and stain with 0.1% coomassie R250 in 40% methonal and 10% acetic acid.

One paper used just 20% trichloroacetic acid to visualize the bands. Another paper used 0.1% coomassie R250 in 20% TCA (destain with 20% TCA).

It seems that my staining method was similar with theirs. I don't understand why I don't get the bands. Please let me know if you have suggestions. Thanks a lot!

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