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I need to calibrate the cDNA amount that i need to use in real time PCR with SYBR Green.
I understand that i need to test several cDNA dilution, to find out the right cDNA amount that is suitable for working with each primer, and to see that the primers behave in a linear way e.g if there are two cDNA concentrations one x4 from the other, than the Ct should be also 4 times different, am i right ?

But how do i know the cDNA amount that i have to dilute after the reverse transcription PCR ?
I've been told that i need to use 100ng, 25ng, 6.25ng, 1.56ng and 0.39ng of cDNA in the calibration (dilutions of X4).

I've heard that maybe it can be calculated based on the RNA amount used in the RT-PCR ?
So if i have 2ul of RNA used in the RT-PCR kit, than i assumed that i have also 2ul cDNA ?
I am using

Once I've done that for a primer and found a workable dilution, can i always use the same cDNA amount each time with that primer for all the cDNA samples that i am using ?

Hi all,

I have cells frozen in FBS+10% DMSO.

Is it possible to extract and to purify RNA from these cells?
Is it necessary to freeze the cells in PBS intead of FBS+DMSO in order to extract RNA ?

I can use Qiagen RNeasy kit, or trisole.

Thanks in advance for the help.

Hi,

I need to count and re-plate cells.

In order to count cells with hemocytometer a serum free medium is needed, so after using trypsin and medium with FBS, I've used a centrifuge to take the medium out and tham put in PBS, and than counted the cells with hemocytometer and trypan blue.

Now, how do i plate the cells after that ?
At this point i have cells with PBS, but if i centrifuge the cells and take out the PBS and than re-suspend the cells in DMEM+FBS, i will change the cells concentration and the cell count will not be correct.

Is it essential to take out the PBS before plating the cells ?
Can i dillute the cells with DMEM+FBS without taking out the PBS ?

I have counted 2000000 cells in 1ml the cells medium using hemocytometer.

how do i load 190micL with 3000 cells (190micL with 3000 cells in each well in a 96 wells plate) ?