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yyc's Content

There have been 6 items by yyc (Search limited from 25-September 19)


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#162748 PCR product sequencing

Posted by yyc on 23 November 2013 - 01:14 PM in Molecular Cloning

Hi,

 

I would like to know if it is possible to sequence direclty a PCR product without first cloning the fragment.

 

Thank you.




#161240 Calcutation question

Posted by yyc on 14 October 2013 - 09:36 AM in Tissue and Cell Culture

Got it!

Thank you very much DRT. Very useful.




#161194 Calcutation question

Posted by yyc on 13 October 2013 - 10:06 AM in Tissue and Cell Culture

Thank you jerryshelly1 and DRT for you explanations and help.

 

DRT, I am quite interested in your calculations of the final volume: What happen then if my stimulators have to be diluted by different factor. For example: stimulator A 1/20; stimulator B 1/100; stimulator C 1/200.

For 1 ml of cell culture. How I calculate the final volume?

 

Sorry, maybe this is quite simple question, but it is something you don't dare to ask and then you acumulate this doubt for long time.

 

Thank you!




#161028 Calcutation question

Posted by yyc on 08 October 2013 - 11:09 AM in Tissue and Cell Culture

Hi,

 

I am a first year PhD student.

I have a calculation question:

 

I have to culture cells in 1 ml and then I have to add some stimulators and enhancers. I have 3 of these stimulators and the final concentration in the cell culture for each stimulator should be: Stimulator A: 1.2 mg/ml; stimulator B: 1 mg/ml; stimulator C: 2 mg/ml.

 

If I make the calcultation for stimulator A (stock concentration is 120 mg/ml), then I have to add 10.16 microlitres, following the equation V1 x C1 = (V2 + V1) x C2:

V1 x 120 mg/ml = (1ml + V1) x 1.2 mg/ml

 

But now I have to add stimulator B (stock concentration 100 mg/ml) and I have a volume of 1011.6 microlitres in the cell culture due to the volume added with stimulator A. Doing the same equation:

V1 x 100 mg/ml = (1.011 ml + V1) x 1 mg/ml

That makes 10.2 microlitres of stimulator B that I have to add.

 

But the problem now is that I have a increase of 10.2 microlitres in the volume of my cell culture due to stimulator B,  therefore stimulator A is not at 1.2 mg/ml anymore due to the increase of volume with stimulator B. And then the same problem when I add stimulator C.

 

Can anybody help me with these calculations?

 

thanks. YYC

 

No need to triple post things - I've deleted your other two identical posts!  Bob.




#160266 Cytokines feeding cell culture

Posted by yyc on 14 September 2013 - 12:24 PM in Cell Biology

Hi everybody,

I need to add cytokines to my cell cultures and in some protocols say I should add certain amount of cytokines per number of cells and in other protocols only refer to cytokines concentration in the culture.

If I consider cytokine concentration it looks it is irrelevant the cell number I have in the culture. But maybe for stimulation with cytokines it is in fact important the amount of cytokines per number of cells.

I would appreciate your comments! thanks!
 



#157402 B cells cultures. Immunoglobuline production

Posted by yyc on 07 July 2013 - 01:58 AM in Cell Biology

Hi,

I am new in the field of cell culture. I am performing an experiment to detect IgE production in the supernatants of isolated and stimulated B lymphocytes. According to my protocol I should take the supernatants from my cell culture (I plate 100.000 B cells in 100 microlitres in 96 well plate) at day 14 in order to analyze the IgE. But at that day the medium is completely yellow.

Does anybody have any experience on measuring IgE in B cell culture supernatants? Is day 14 already too late? The medium is yellow so, I am afraid most of the cells are already dead at that time.

I would appreciate you share your experiences with me.

Thank you,

YM




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