thekid

Hello All,

              Am currently working with immune response among vaccinated people and would like to know if anyone knows how to calculate the take of the vaccine, its also called vaccine take.  I have been trying to get some form of literature supporting these words and would be great if anyone here knows the term.



thekid

Hello,

     I recently did a protein estimation by BCA assay which had a standard protein concentration starting from 25ug/ml - 2000ug/ml.

   The spectrophotometer that I used for the assay does not give OD's above 1.0 and hence of the 6 diluted standards I prepared I was able to use only 3 of them from 25ug/ml - 100ug/ml after which the OD's was above 1.0 and the reader was not able to give any values.

My question is : Is it valid to dilute the standards that you have made from 200ug/ml - 2000ug/ml by 1:1 and than take the OD values and multiply it by the dilution factor (0.5 in this case). And than use the ODs obtained here and before (25ug/ml - 100ug/ml) and construct the graph to determine the concentration of the unknown protein sample?


If this is a valid assay, is there any reference that you can suggest for this, and if you think it is wrong to do this please provide an explanation for the same.


regards,
thekid

Hello,

    Is there anyway I can open the ABI 7500 files in a macbook, the ABI does not supply the software that works on Mac, does anyone know of any free software that will open the documents in Mac.


Thank You,
thekid

Hello,

        I am trying to standardize a taqman assay using a probe of 24mer with Fam as reporter and Tamra as the quencher, the probe is from SIGMA and is at stock concentration of 100UM made with TE buffer.  The Calculated Tm of the probe is 65.3C and those of the primers are 63C. Am using the ABI Universal Taqman reagent with UNG for the assay. The conditions are 50C for 2 mins, 95C for 10mins and 35 cycles of 95C for 15sec and 60C for 1min.

I use diluted plasmids from copy number starting from 10 power 7 to 10 power 2 in a ABI 7500 machine. I have previously checked all the diluted plasmids and primers with SYBR green and they have given me r2 of 0.999 and efficiency of 98.9%. But when I use the same set of primers and diluted plasmids for the Taqman assay it does not work. There is no amplification see at all. When I run the products on a gel I see clear bands without any primer dimer or non specific bands.

I am using :

8]Taqman MasterMix 2x         12.5ul

    primer (20pmoles/ul)           1.0ul
    primer  (20pmoles/ul)          1.0ul
                        probe (20uM)                       0.2ul
    RNase-free H2O                  8.3 ul
                                                                    23.0ul
                       Plasmid                                 2.0ul
I have played with increasing and decreasing the probe concentration, anneling temperature went down to as low as 50C and as high as 62C with 2 degree increments every tube had good bands as observed on a gel but no amplification was seen on the plot.

Is it a problem with the probe?



thekid