RT PCR setup -384 Well
Preparing samples:
1) From an RT using 500ng RNA we usually make our cDNA
dilutions in 96 well plates.
2) Plan sample order on spreadsheet
3) Make 1:75 dilution of samples and negative controls and a 1:50
dilution for the first point of the titration curve (SC). SC can be
made from previously prepared samples or a pool of your controls.
4) Spin down plate.
5) Prepare a 6 point titration curve of serial dilutions in tubes as
follows.
SC = 1:50 dilution as above > S2 = 1;100 H20 > S3 = 1:200 etc……..
6) Take care to vortex and spin down in between making each
standard dilution.
7) Add full volume to cDNA plate in appropriate wells as per
spreadsheet.
8) Put on temporary lid and spin down before adding to 384 well
plate.
Preparing 10X primers:
1) Label a 1.5ml eppendorf by gene name and F/R/P
(Forward/Reverse/Probe) or F/R if it is a SYBR set.
2) For a taqman set 10x:
617ul H20
20ul 100uM F
20ul 100uM R
10ul 100uM Probe
For a SYBR set 10x:
627ul H20
20ul 100uM F
20ul 100uM R
3) Prepare the mix for gene of interest taking care to make enough for
duplicates of your samples, the standards, -VEs and a few extra
samples:
For a taqman or SYBR set:
(1x)
2x Mastermix 6.5ul
F/R/P
1.3ul
H20
0.2ul
4) Make a note of the order in which your samples and SC will be
loaded into a 384 well plate.
5) Using the multichannel pipette, load 5ul cDNA into appropriate
wells of 384 well plate in duplicate.
6) Use a clean tip for each sample. If you use a new tip box it will
help you keep track of what you have done.
7) Put on temporary lid and spin down.
8) Using electronic pipette add 8ul mix to each well.
9) Apply optical lid and spin plate down, store in -20C or load
immediately.