qPCR

In this test we are going to determine how much mouse and bacterial DNA is in the sample
you have prepared. We have a primer set to detect a mouse sequence (in the TAT gene) and
also for Bacteroides.

1. Prepare DNA dilution cure

The relative concentrations are:

1. 1 use neat in the well
2. 1/10
3. 1/100
4. 1/1000
5. 1/10000
6. 1/100000

2. Make 6x DNA solutions, this well prepare a relative standard curve

3. Prepare 20 ul of each standard in water.

Label 6 tubes

Sample DNA Water

1 your DNA sample Use 2 ul per well
2 2 of sample 1 18
3 2 of sample 2 18
4 2 of sample 3 18
5 2 of sample 4 18
6 2 of sample 5 18

4. Add 2ul of each sample to the wells as show below

5. Make up a qPCR master mix for each primer as shown below. Label2 tubes

1.TAT
2. Beta
TAT target BAC target
Reaction vol 20 uL Reaction vol 20 uL
1 sample 8 samples 1 sample 8 samples
Master mix (x2) 10 80 Master mix (x2) 10 80

Forward primer 0.4 3.2 Forward primer 0.4 3.2

Reverse Primer 0.4 3.2 Reverse Primer 0.4 3.2
Water 7.2 57.6 Water 7.2 57.6

final vol uL 18 144 final vol uL 18 144

(2x master mix is GoTaqQPCR)

Add 18 uL of your master mix to the correct well, see below

Plate layout example

TAT BAC TAT BAC TAT BAC
1 2 3 4 5 6 7 8 8 10 11 12
A Sample1 Sample1 Sample1 Sample1 Sample1 Sample1
B Sample2 Sample2 Sample2 Sample2 Sample2 Sample2
C Sample3 Sample3 Sample3 Sample3 Sample3 Sample3
D Sample4 Sample4 Sample4 Sample4 Sample4 Sample4
E Sample5 Sample5 Sample5 Sample5 Sample5 Sample5
F Sample6 Sample6 Sample6 Sample6 Sample6 Sample6
G water water water water water water
H
Student 1 Student 2 Student3