We are planning to perform qPCR in 384 or 1536-multiwell plates.
However, the total reaction volume seems to be too small (2.5-100nl) to carry out qPCR. Practically, It seems to be very difficult for doing qPCR with many different genes with the same gDNA samples simutaneously.
In this case, what is the best method for adding the primers and other reaction mixture including template and cybergreen dye? should each primers be added and dried in advance before adding others or not?
Does anybody know the best method (or sequence) for adding the components for qPCR in this situation?
Dont you suppose to use between 0.2-2.0 ul in a 1536 weel plate? my qpcrs i always use 10ul reaction.
Hi, Pombo,
Yes, we are planning to do in less than 2ul in multi well plates (384 or 1536 -weell).
In this case, what is the best method for adding the primers and other mixture components for qPCR of many different genes simultaneously?
what i would do is a master mix of the components that are enough for the number of samples( do always a bit more). do a master mix to each gene. add the master mix to all wells and then the sample.add a control (blanks). never used such small amounts on my qpcrs.
"the total reaction volume seems to be too small (2.5-100nl) to carry out qPCR"
(2.5-100nl) or (2.5-100ul) you meant microliter here?
I have not yet used 1536-well plates. As you suggested, it may be better to add primers and dry before adding a master mix containing cDNA. Origene provides cancer tissue cDNA array dried in a 384-well plate to measure a particular gene expression in various cancer tissues. Similarly, Qiagen provides RT2 Profile PCR array in a 384-well format (the primer pairs of different genes aliquoted individually in each well and dried) for analysing the expression of a focused panel of genes in a particular tissue.
Hi, Sandra
I meant 2.5-100nl, not ul.
But, my major question is 'What is the best method for adding different primers and other mixtures in doing qPCR in multi-well plates simultanelusly.
As Kanamalapudi answered above, adding primers and drying them in advance is considered more appropriate for HTS of many different genes in the same plates.However, in this case, I have another concern that primer drying may affect the quality of qPCR reaction compared to non-drying method.
How do you think about this?
Hi, Kanamalapudi
Thanks for your answers. adding primers and drying them in advance is considered more appropriate for HTS of many different genes in the same plates.However, in this case, I still have another concern that primer drying may affect the quality of qPCR reaction compared to non-drying method.
How was the experiments in Origene's 384-well plates?
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