High-Throughput qPCR method on
Biomek FX with 384-well plates
This method works to combine a perfect matrix of all samples with all assay mixes. So, for example, 8
samples with 12 assays (genes, primer mixes…) will result in 96 individual combinations. Multiply this
by the well replication (usually 3) and you have the number of wells used on the 384-well run plate
(288 wells in this case). You need to decide on your plate configuration in terms of numbers of, and
which, samples to run on each plate and the number of genes or assays. You are encouraged to try
to maximize sample-to-sample comparisons and try to run all samples together on each 384-well
plate and run the different assays on different plates.
Terminology:
Sample – individual sample to be analysed (not including any technical or well replication). The set of
samples should include all your cDNA/DNAs and also standards, calibrators, controls (-ve and +ve)
Assay – this is what you are measuring in your samples – usually this means gene (for gene
expression studies) but also could be SNP/copy number assay, or genetic marker for metagenomics
projects. It usually refers to an ‘assay mastermix’, containing all enzymes for amplification, primer
pairs, probes, dNTPs, MgCl2 etc.
Pattern – arrangement of wells used on a plate
Information required: please fill in the following values
Data
Description
Number of samples
The total number of samples in
your study, including standards,
controls (-ve and +ve), etc.
Volume of your samples – it’s
good if these are all the same, but
can be variable
The total number of assays or
genes you are interested in for
the whole study
The number of assays you will run
on each plate
Sample stock
volume(s)
Total Number of
assays
Number of assays per
plate
Wells per assay
Assay stock volume
Total Number of wells
per run plate
How many wells contain each
assay you are running? Usually 12
Volume of liquid in each assay
well
Total number of wells containing
samples in your 384-well run
plate
Value
Variable name (in
method)
no_of_samples
sample_stock
n/a
no_of_assays
wells_per_assay
assay_stock
n/a
Robot Method
The samples and assays are stored in 96-well plates of various types. These are predefined in the
robot method
Defined plates:
Plate name
samples
Wells
96
Description
plate containing your samples.
Position
assays
96
plate containing all your assay mastermixes
run
384
Destination plate for setting up your qPCR
reactions
Defined Patterns:
You need to indicate which wells on each plate contain samples and assays, and where to combine
them on the run plate.
To work out the run plate pattern, first you need to you have to work out how many reaction wells
there will be on your destination (run) plate.
Formula
No. of samples
example
*
28
No. of assays
per plate
4
*
well replicates =
No. of reaction wells
3
336
Your data
Defined Pattern name
sample_wells
assay_wells
run_wells
Description
Which wells contain samples on ‘samples’ plate
Which wells contain assay mastermix on ‘assays’plate
Which wells on your 384-well ‘run’ plate will contain samples (=
reaction wells)
Table 1 Descriptions of different patterns used in method
Please indicate which wells are to be used on the patterns below
sample_wells
1
A
B
C
D
E
F
G
H
2
3
4
5
6
7
8
9
10
11
12
assay_wells
1
2
3
4
5
6
7
8
9
10
11
12
A
B
C
D
E
F
G
H
The run_wells plate pattern is defined by the robot – you don’t have to fill this one in yourself
run_wells
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Odd numbers
If you have an odd number of samples, you may need to add an extra NTC sample to fit with the
standard ‘square’ patterns the robot uses. Please discuss with Genomics staff.
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