PCR & Western Blot sample preperation - (Jan/14/2015 )

Hello,

I am VERY new at leaning PCR and western blotting. I have jumped in at many different times during both processes and am confused as to what needs to be done when and for which. My questions are:

1) In general, how would you prepare a sample of cancer cells to perform RT-PCR and then qPCR?

2)In general, how would you prepare a sample of cancer cells to run a western blot?

I am especially confused as to when I have sonicate the samples, when to use loading buffer, DTT, and when RNA isolation is needed.

 I know these questions may require long answers, but it would help me immensely.

Thanks!

you would isolate rna prior to reverse transcription-pcr

western blot is used to look at specific protein(s). the sample would be solubilized with loading buffer (and, usually, reducing agent (dtt or mercaptoethanol))

you might sonicate the cells to disrupt the cell membranes rather than use (or use in conjunction with) detergents. keep in mind that sonication in the presence of detergents will cause significant foaming, you should sonicate prior to addition of detergent.

mdfenko on Thu Jan 15 12:36:28 2015 said:

you would isolate rna prior to reverse transcription-pcr

 

western blot is used to look at specific protein(s). the sample would be solubilized with loading buffer (and, usually, reducing agent (dtt or mercaptoethanol))

 

you might sonicate the cells to disrupt the cell membranes rather than use (or use in conjunction with) detergents. keep in mind that sonication in the presence of detergents will cause significant foaming, you should sonicate prior to addition of detergent.

This is very helpful! Thank you!!