FLUIDIGM EP1 PROTOCOL
1. Pre-Amplification Plate
Pooled Assays Mix
1)
Primers (200µM each, Rev and Fwd
separate)
2mM Tris/Water
Total
2.5µL
each
520 µL
480 µL
520 µL
1000µL
2) Dilute the assay mix 1:10
Pre-Amplification Cocktail (µL)
Per
Sample
2x Qiagen Master Mix
3.5
Pooled Assay Mix
0.875
MQ-H20
0.625
DNA
2
Total
7
96 wells plate
371
92.75
66.25
Pre-Amp Thermal Cycle Profile
95°C
15 min
Hold
95°C
15 sec
14 cycles
60°C
4 min
4°C
∞
Add 135 µL H20 or 1x TE to each sample
Store at 4⁰ until genotyped. Store at -20⁰ after genotyped.
2. Running the Chip
Preparation
Assay Mix
Use and label 1.5 µL Eppendorf Tube, don’t dispense yet.
ASSAYS Preparation (µL)
SNP assay
1x
1.25
106x
2x assay loading reagent
ROX (50x)
diH20
Total
2.5
0.25
1
5
282.67
28.267
113.07
PREPARE ASSAY PLATE
-
Vortex and spin down Assay Mix
Spin down Assay Plate
Dispense 4µL of Assay Mix into each well of 96 well plate (use 300
µL Eppendorf tips)
Add 1.3 µL of Assay (use multi-channel with red tips)
SPIN plate
Sample Mix
Use and label 1.5 µL Eppendorf Tube, don’t dispense yet.
SAMPLE Preparation (µL)
1x
106x
TaqMan Universal Master Mix
2.5
353.33
0.25
35.33
20x GT Sample loading reagent
AmpliTaq Gold DNA Polymerase
0.05
7.067
diH20
0.2
28.267
Pre-amped DNA
2.67
Total
6.67
PREPARE SAMPLE PLATE
-
Vortex and spin down Sample Mix
Spin down Sample Plate (Pre-amp plate)
Dispense 4µL of Sample Mix into each well of 96 well plate (use 300
µL Eppendorf tips)
Add 2.3 µL of each Sample (use multi-channel with red tips)
SPIN plate
PRIME THE CHIP
Inject Control Line Fluid (in syringes) into both wells of chip
o
Hold the chip at a 45-degree angle
o
Make sure to depress valve with end of syringe
o
dispense entire volume
You can remove the blue film underneath the chip at this time
Place the chip into IFC controller, run the Prime (138x) script
Note A1 well placement
Tips:
-Prepare mix before priming chip.
-Prepare plate while chip is priming
-ASSAY on the LEFT side of the chip (with A1 notch)
-SAMPLE on the RIGHT side of the chip (without A1 notch)
LOAD THE CHIP
-
Remove chip from IFC Controller (when script has finished)
Add 4 µL of each ASSAY to LEFT SIDE of chip (with A1 notch)
Add 5 µL of each SAMPLE to RIGHT SIDE of chip (without A1
notch)
Return chip to IFC Controller
Run Load Mix (138x) script
2 negative samples – use H20
RUN PCR
-
When Load Mix has finished, transfer plate to PCR machine
Turn on main power (in back on the right)
Turn on vacuum power (on the front) – should drop below -80
Press ENTER (“start”)
Press ENTER again (“PCRGTM96”)
READING THE CHIP
-
Shut off vacuum when the PCR is finished and remove chip
Use scotch tape to remove any dust on the chip surface
Load into EP1 and follow instructions
Fluidigm SNP Genotyping Analysis
-
Set up Assay & Sample Plate
-
Mark NTC for the two negative controls
Last Updated: 3/23/2011