GoldenGate Genotyping Assay Experienced User Card, Manual Protocol for Universal Array Matrix (15005074 A)

GoldenGate Genotyping Assay Experienced User Card, Manual Protocol for Universal Array Matrix (15005074 A)
GoldenGate® Genotyping Assay, Manual
Experienced User Card (Universal Array Matrix)
Day 1
Day 2
Day 3
Make QDNA
Add MEL
Wash UAM
45 min/2 plates
60 min/3 plates
Reagents: Lambda DNA,
PicoGreen dsDNA, 1X TE
Output: Standard and
Sample QDNA Plates
Incubation: 15 min
Reagents: MEL, AM1, UB1
Output: ASE Plate
Hands-On: 30 min/UAM
Reagents: 95% Ethanol,
2-butanol, IS1, UB2
Output: Universal Array
Matrix
Make PCR
Reagents: MMP, UDG,
DNA Polymerase
Output: PCR Plate
Read QDNA
Fluorometer: 5 min/plate
Input: Standard and
Sample QDNA Plates
Output: File identifying
quantity of DNA in the
sample plate
Image UAM
BeadArray Reader Scan
Time: 1.5 hrs/UAM
Output
Image and Data Files
Inoc PCR
Reagents: UB1, IP1
Output: PCR Plate
Cycle PCR
Make SUD
(Optional Batch)
Make MUD
Incubation: 30 min
Reagents: MM1
Output: MUD Plate
Incubation: 30 min
Reagents: MS1
Output: SUD Plate
Cycle: 2 hours 45 min
Hold overnight at 10ºC
Output: PCR Plate
Bind PCR
Precip SUD
Precip MUD
Dry: 60 min
Reagents: 2-propanol,
PS1
Output: SUD Plate
Dry: 60 min
Reagents: 2-propanol,
PS1
Output: MUN Plate
Resuspend SUD
Resuspend MUN
Reagents: RS1
Output: SUD Plate
Reagents: RS1
Output: MUN Plate
Incubation: 60 min
Reagents: MPB,
Sec-butanol, 70% EtOH
Output: PCR Plate
Make HYB Plate
for UAM
Hands-On: 45 min
Reagents: UB2, MH1,
0.1N NaOH
Output: HYB Plate
Hyb UAM
Make SUD ASE
(Optional Batch)
Incubation: 2 or 16 hours
Reagents: OPA, OB1
Output: ASE Plate
Hands-On: 30 min
Incubation: One 30 min/
one 14 hrs
Reagents: UB2,
0.1N NaOH
Output: HYB Plate
UAM
Optional
Pre-PCR
Post-PCR
Optional stop,
cold storage
Long
incubation
Fill in the lab tracking
form and the sample
sheet as you perform
the assay
Catalog # GT-901-1001
Part # 15005074 Rev A
Page 1 of 38
GoldenGate® Genotyping Assay, Manual (Pre-PCR)
Experienced User Card (Universal Array Matrix)
Make QDNA
Plate
(Optional)
Estimated Time
Consumables
Preparation
Steps
This process uses the Quant-iT PicoGreen dsDNA quantitation reagent
to quantitate double-stranded DNA samples for the GoldenGate
Genotyping Assay for VeraCode.
Hands-on: 20 minutes per plate, plus 10 minutes to prepare the PicoGreen
Item
Quantity
Storage
Supplied By
PicoGreen dsDNA
quantitation reagent
See instructions
-20ºC
User
1X TE
See instructions
Room
temperature
User
Lambda DNA
See instructions
-20ºC
User
96-well 0.65 ml microplate
(MIDI)
1 per 96 samples
User
Fluotrac 200 (96-well black
flat-bottom) plate
2 per 96 samples
User
[ ]
Remove PicoGreen reagent from freezer and thaw at room temperature
for 60 minutes in a light-impermeable container.
[ ]
Label a 96-well MIDI plate “Standard QDNA.”
[ ]
Label a 96-well black flat-bottom plate “Standard QDNA.”
[ ]
Label a 96-well black flat-bottom plate “Sample QDNA.”
Make Standard QDNA MIDI Plate
[ ] 1. Place stock Lambda DNA in well A1 of the Standard QDNA MIDI plate
and dilute it to 75 ng/μl in a final volume of 233.3 μl.
[ ] 2. Add 66.7 μl 1X TE to well B1 of the same plate.
[ ] 3. Add 100 μl 1X TE to wells C, D, E, F, G, and H of column 1 of the same
plate.
[ ] 4. Pipette the contents of A1 up and down 10 times to mix.
[ ] 5. Transfer 133.3 μl of Lambda DNA from well A1 into well B1, and then
pipette the contents of well B1 up and down 10 times.
[ ] 6. Change pipette tips. Transfer 100 μl from well B1 into well C1, and then
pipette the contents of well C1 up and down 10 times.
[ ] 7. Change pipette tips. Transfer 100 μl from well C1 into well D1, and then
pipette the contents of well D1 up and down 10 times.
[ ] 8. Change pipette tips. Transfer 100 μl from well D1 into well E1, and then
pipette the contents of well E1 up and down 10 times.
[ ] 9. Change pipette tips. Transfer 100 μl from well E1 into well F1, and then
pipette mix the contents of well F1 up and down 10 times.
Catalog # GT-901-1001
Part # 15005074 Rev A
Page 3 of 38
GoldenGate® Genotyping Assay, Manual (Pre-PCR)
Experienced User Card (Universal Array Matrix)
[ ] 10. Change pipette tips. Transfer 100 μl from well F1 into well G1, and then
pipette the contents of well G1 up and down 10 times.
[ ] 11. Do not transfer solution from well G1 to well H1. Well H1 serves as the
blank 0 ng/μl Lambda DNA.
[ ] 12. Cover the plate with a cap mat.
[ ] 13. Do one of the following:
•
Proceed to Prepare Standard QDNA Fluotrac Plate with PicoGreen
Dilution.
•
Store the plate at 4°C for future use.
Prepare Standard QDNA Fluotrac Plate with PicoGreen Dilution
[ ] 1. Prepare a 1:200 dilution of PicoGreen to 1X TE, using the kit supplies
and a sterile 100 ml plastic container wrapped in aluminum foil.
[ ] 2. Cap the sterile plastic container and vortex to mix.
[ ] 3. Pour the PicoGreen/1X TE dilution into a sterile reservoir.
[ ] 4. Using an 8-channel pipette, transfer 195 μl PicoGreen/1X TE dilution into
each well of columns 1 and 2 of the Standard QDNA Fluotrac plate.
[ ] 5. Add 2 μl of each stock Lambda DNA dilution from column 1 of the
original Standard QDNA MIDI plate into the corresponding wells of
columns 1 and 2 in the Standard QDNA Fluotrac plate.
[ ] 6. Pipette mix the contents of the new Standard QDNA plate.
[ ] 7. Immediately cover the plate with an aluminum adhesive seal.
Prepare Sample QDNA Fluotrac plate with PicoGreen and DNA
[ ] 1. Transfer 195 μl of the PicoGreen/1X TE dilution that you made earlier
into each well of the new black flat-bottom plate labelled “Sample
QDNA”.
[ ] 2. Add 2 μl sample DNA to each well of the Sample QDNA plate.
[ ] 3. Pipette mix the contents of the Sample QDNA plate.
[ ] 4. Immediately cover the new plate with an aluminum adhesive seal.
[ ] 5. Proceed to Read QDNA Plate (Optional).
Catalog # GT-901-1001
Part # 15005074 Rev A
Page 4 of 38
GoldenGate® Genotyping Assay, Manual (Pre-PCR)
Experienced User Card (Universal Array Matrix)
Read QDNA
Plate
(Optional)
Estimated Time
Steps
This process uses the Gemini XS or XPS Spectrofluorometer to provide
DNA-specific quantitation. Illumina recommends using a fluorometer,
because fluorometry provides DNA-specific quantitation, whereas
spectrophotometry may also measure RNA and yield values that are
too high.
Fluorometer: 5 minutes per plate
[ ] 1. Turn on the fluorometer.
[ ] 2. At the PC, open the SoftMax Pro program.
[ ] 3. Load the Illumina QDNA.ppr file.
[ ] 4. Select Assays | Nucleic Acids | Illumina QDNA.
[ ] 5. Place the Standard QDNA Plate into the fluorometer loading rack with
well A1 in the upper-left corner.
[ ] 6. Click the blue arrow next to Lambda Standard.
[ ] 7. Click Read in the SoftMax Pro interface.
[ ] 8. Remove the Standard QDNA Plate from the drawer.
[ ] 9. Click the blue arrow next to Standard Curve to view the standard curve
graph.If the standard curve is acceptable, continue with the sample
plate. Otherwise, click Standard Curve again.
[ ] 10. Place the Sample QDNA plate in the reader with well A1 in the upper left
corner.
[ ] 11. Click the blue arrow next to QDNA#1 and click Read.
[ ] 12. Remove the plate from the drawer.
[ ] 13. Repeat steps 11 through 13 for all sample plates that you want to
quantitate.
[ ] 14. Once all plates have been read, click File | Save to save the output data
file (*.pda).
[ ] 15. Proceed to Make SUD Plate.
Catalog # GT-901-1001
Part # 15005074 Rev A
Page 5 of 38
GoldenGate® Genotyping Assay, Manual (Pre-PCR)
Experienced User Card (Universal Array Matrix)
Make SUD
Plate
Estimated Time
Consumables
Preparation
Steps
This process activates sufficient DNA of each individual sample to be
used once in the GoldenGate Genotyping Assay for VeraCode.
Hands-on: ~15 minutes
Incubation: 30 minutes
Item
Quantity
Storage
Supplied By
10 mM Tris-HCl pH 8.0, 1 mM See instructions
EDTA (TE)
Room
temperature
User
MS1 reagent
1 tube per SUD
plate
-20ºC
Illumina
DNA samples and controls
96 or 384
-20ºC
User
96-well 0.2 ml skirted
microplate
1 per sample plate
User
[ ]
Preheat the heat block to 95°C.
[ ]
Turn on the heat sealer to preheat it. Allow 15 minutes.
[ ]
Thaw the MS1 reagent tube to room temperature. Vortex to mix the
contents, and pour the entire tube into a new, non-sterile reservoir.
[ ]
Thaw the DNA samples and controls to room temperature and vortex to
mix the contents.
[ ]
Apply a SUD barcode label to a new microplate.
[ ] 1. Normalize DNA samples to 50 ng/μl with 10 mM Tris-HCl pH 8.0, 1 mM
EDTA.
[ ] 2. Add 5 μl MS1 reagent to each well of the SUD plate.
[ ] 3. Using an 8-channel pipette, transfer 5 μl normalized DNA sample to each
well of the SUD plate. Change tips between column dispenses.
[ ] 4. Apply a microplate foil heat seal to the SUD plate and seal it with the
heat sealer (3 seconds).
[ ] 5. Pulse centrifuge the SUD plate to 250 xg.
[ ] 6. Vortex at 2300 rpm for 20 seconds.
[ ] 7. Pulse centrifuge to 250 xg.
[ ] 8. Place the SUD plate in the preheated heat block and close the lid.
[ ] 9. Incubate the SUD plate at 95°C for exactly 30 minutes.
[ ] 10. Pulse centrifuge the plate to 250 xg.
[ ] 11. If you plan to perform the Make ASE protocol today, then immediately
set the heat block to 70°C.
[ ] 12. Proceed to Precip SUD Plate.
Catalog # GT-901-1001
Part # 15005074 Rev A
Page 7 of 38
GoldenGate® Genotyping Assay, Manual (Pre-PCR)
Experienced User Card (Universal Array Matrix)
Precip SUD
Plate
Estimated Time
Consumables
Preparation
Steps
In this process, PS1 and 2-propanol are added to the SUD plate to
precipitate the DNA and remove excess DNA activation reagent MS1.
Hands-on: ~30 minutes
Drying: 15 minutes
Item
Quantity
Storage
Supplied By
PS1 reagent
Bottle
4ºC
Illumina
2-propanol
Bottle
Room
temperature
User
[ ]
Pour 1 ml PS1 into a reagent reservoir.
[ ]
Pour 2 ml 2-propanol into a second reservoir.
[ ] 1. Remove the heat seal from the heated SUD plate.
[ ] 2. Add 5 μl PS1 reagent to each well of the SUD plate.
[ ] 3. Seal the SUD plate with clear adhesive film.
[ ] 4. Pulse centrifuge the plate to 250 xg.
[ ] 5. Vortex at 2300 rpm for 20 seconds or until the solution is uniformly blue.
[ ] 6. Remove the film and add 15 μl 2-propanol to each well of the SUD plate.
[ ] 7. Seal the SUD plate with clear adhesive film.
[ ] 8. Vortex at 1600 rpm for 20 seconds or until the solution is uniformly blue.
[ ] 9. Centrifuge the sealed SUD plate to 3000 xg for 20 minutes. A faint blue
pellet should be at the bottom of each well.
Perform the next step immediately to avoid dislodging the activated
DNA pellets. If any delay occurs, recentrifuge to 3000 xg for 10 minutes
before proceeding.
[ ] 10. Remove the SUD plate seal and decant the supernatant by inverting the
SUD plate and smacking it down onto an absorbent pad.
CAUTION
Do not tilt the plate, as this can cause cross-contamination
between wells. Tap the plate firmly enough to decant all
the supernatant; tapping lightly will not work as well.
[ ] 11. Tap the inverted plate onto the pad to blot excess supernatant.
[ ] 12. Place the inverted SUD plate on an absorbent pad and centrifuge to 8 xg
for 1 minute.
Catalog # GT-901-1001
Part # 15005074 Rev A
Page 9 of 38
GoldenGate® Genotyping Assay, Manual (Pre-PCR)
Experienced User Card (Universal Array Matrix)
WARNING
Do not spin the inverted plate to more than 8 xg, or the
sample will be lost!
[ ] 13. Set the plate upright and allow it to dry at room temperature for
15 minutes.
[ ] 14. Do one of the following:
Catalog # GT-901-1001
Part # 15005074 Rev A
•
Proceed to Resuspend SUD Plate.
•
Seal the plate with adhesive film and store at -20ºC for up to
24 hours.
Page 10 of 38
GoldenGate® Genotyping Assay, Manual (Pre-PCR)
Experienced User Card (Universal Array Matrix)
Resuspend
SUD Plate
Estimated Time
Consumables
Preparation
Steps
In this process, RS1 is added to the SUD plate to resuspend the DNA.
Hands-on: ~15 minutes
Item
Quantity
Storage
Supplied By
RS1 reagent
Bottle
Room
temperature
Illumina
[ ]
Pour 1.2 ml RS1 into a reagent reservoir.
[ ] 1. Add 10 μl RS1 reagent to each well of the SUD plate.
[ ] 2. Seal the SUD plate with microplate clear adhesive film.
[ ] 3. Pulse centrifuge to 250 xg.
[ ] 4. Vortex at 2300 rpm for 1 minute or until the blue pellet is completely
dissolved.
[ ] 5. Do one of the following:
Catalog # GT-901-1001
Part # 15005074 Rev A
•
Proceed immediately to Make ASE Plate.
•
Store the SUD plate at 4°C overnight or at -20ºC for one month.
Page 11 of 38
GoldenGate® Genotyping Assay, Manual (Pre-PCR)
Experienced User Card (Universal Array Matrix)
Make MUD
Estimated Time
Consumables
This process activates sufficient DNA of each individual sample to be
used at least SIX TIMES in the GoldenGate genotyping assay.This
process requires 2 μg of each DNA sample at 50 ng/μg. The plate is
incubated at 95ºC for 30 minutes to activate the genomic DNA. Fill in
the lab tracking form as you work.
Robot: 20 minutes per plate
Incubation: 30 minutes
Item
Quantity
Storage
Supplied By
MM1 reagent
1 tube per MUD
plate
-20ºC
Illumina
10 mM Tris pH 8.0/1 mM
EDTA
See instructions
Room
temperature
User
Genomic DNA Platea
Up to 3 plates
MIDI: > 57 μl per
sample well
TCY: > 47 μl per
sample well
Room
temperature
User
96-well 0.2 ml skirted
microplate (TCY)
1 per 96 samples
User
a. Thawed, normalized to 50 ng/μl, diluted in 10 mM Tris pH 8.0/1 mM EDTA, and
quantitated using the PicoGreen method.
Preparation
Steps
[ ]
Preheat the heat block to 95ºC (one for each MUD plate). Allow
20 minutes for it to equilibrate.
[ ]
Turn on the heat sealer to preheat it.
[ ]
Thaw each MM1 tube to room temperature. Vortex briefly to mix.
[ ]
Prepare the robot for use.
[ ]
Apply a MUD barcode label to each new 96-well TCY microplate.
[ ]
Pour MM1 tube contents into a sterile trough.
[ ] 1. Add 40 μL MM1 reagent to each well of the GS#-MUD plate.
[ ] 2. Add 40 μL normalized DNA sample to each well of the MUD plate.
[ ] 3. Pipette mix DNA sample and MM1 reagent in the MUD plate.
[ ] 4. Apply the microplate foil heat seal to the MUD plate and heat-seal it with
the heat sealer.
[ ] 5. Pulse centrifuge sealed plate to 250 xg.
[ ] 6. Heat MUD plate at 95°C for 30 minutes in the preheated heat block.
[ ] 7. Using the heat block cover, cover the MUD plate to reduce condensation
on the plate seal.
[ ] 8. Remove the MUD plate from the heat block and pulse centrifuge to
250 xg to remove condensation from the walls of each well.
[ ] 9. Proceed to Precip MUD.
Catalog # GT-901-1001
Part # 15005074 Rev A
Page 13 of 38
GoldenGate® Genotyping Assay, Manual (Pre-PCR)
Experienced User Card (Universal Array Matrix)
Precip MUD
Estimated Time
Consumables
Preparation
Steps
In this process, activated DNA from the MUD plate is transferred to the
multi-use nucleic acid (MUN) plate for precipitation. The PS1 reagent
and 2-propanol are added to the MUN plate to precipitate the DNA. Fill
in the lab tracking form as you work.
Hands-on: ~30 minutes
Incubation: 1 hour
Item
Quantity
Storage
Supplied By
PS1 reagent
Bottle
4ºC
Illumina
2-propanol
Bottle
Room
temperature
User
0.8 ml, V-bottom, deep well
MIDI plate
1 per MUD plate
User
[ ]
Bring all reagents and MUD plate(s) to room temperature.
[ ]
Pour 5 mL GS#-PS1 into a new sterile trough.
[ ]
Pour 13 mL 2-propanol into another sterile trough.
[ ]
Apply a MUN barcode to each new MIDI plate.
[ ] 1. Carefully remove heat seal from heated MUD plate, taking care to avoid
splashing from the wells (see optional Foil Stripper, Materials, UserSupplied on page 2-9).
[ ] 2. Add 40 μL PS1 reagent to each well of the MUN plate.
[ ] 3. Transfer the entire contents (80 μL) from each well of the heated MUD
plate to the corresponding well of the MUN plate.
[ ] 4. Using the cap mat, seal the MUN plate.
[ ] 5. Pulse centrifuge to 250 xg to collect the contents to the bottom of the
wells.
[ ] 6. Vortex at 2000 rpm for 20 seconds.
[ ] 7. Pulse centrifuge to 250 xg.
[ ] 8. Remove the cap mat and add 120 μL 2-propanol to each well of the
MUN plate.
[ ] 9. Using the cap mat, seal the MUN plate and vortex at 1600 rpm for 20
seconds.
[ ] 10. Centrifuge sealed MUN plate at 3000 xg for 20 minutes.
[ ] 11. Remove MUN plate from centrifuge.
[ ] 12. Remove cap mat from MUN plate.
[ ] 13. Decant supernatant by inverting the MUN plate over an absorbent pad.
Press inverted plate onto absorbent pad to blot off excess supernatant.
[ ] 14. Place MUN plate inverted on an absorbent pad and centrifuge at 8 xg for
1 minute.
Catalog # GT-901-1001
Part # 15005074 Rev A
Page 15 of 38
GoldenGate® Genotyping Assay, Manual (Pre-PCR)
Experienced User Card (Universal Array Matrix)
[ ] 15. Remove MUN plate from centrifuge and allow to dry at room
temperature for 15 minutes.
[ ] 16. Proceed to Resuspend MUN.
Catalog # GT-901-1001
Part # 15005074 Rev A
Page 16 of 38
GoldenGate® Genotyping Assay, Manual (Pre-PCR)
Experienced User Card (Universal Array Matrix)
Resuspend
MUN
Estimated Time
Consumables
Preparation
Steps
In this process, RS1 reagent is added to the multi-use DNA (MUN) plate
to resuspend the sample. One MUN plate can inoculate up to six ASE
plates. Fill in the lab tracking form as you work.
Hands-on: ~30 minutes
Item
Quantity
Storage
Supplied By
RS1 reagent
Bottle
Room
temperature
Illumina
[ ]
Pour 12 mL RS1 into a sterile trough.
[ ] 1. Add 100 μL RS1 reagent to each well of the MUN plate.
[ ] 2. Using the cap mat, seal MUN plate and vortex at 2000 rpm for 1 minute
to resuspend the blue pellet.
[ ] 3. Verify that the blue pellet in each well of the MUN plate has been
dissolved back into solution. If not, repeat step 2.
[ ] 4. MUN sample plate activation is complete. Either seal and store at 4°C, or
proceed immediately to Make ASE Plate.
NOTE
For long-term storage, the activated DNA may be frozen at
-20°C. If the activated DNA is stored frozen, thaw
completely and vortex to mix contents before use in assay.
[ ] 5. Proceed to Make ASE Plate.
Catalog # GT-901-1001
Part # 15005074 Rev A
Page 17 of 38
GoldenGate® Genotyping Assay, Manual (Pre-PCR)
Experienced User Card (Universal Array Matrix)
Make ASE
Plate
Estimated Time
Consumables
Preparation
Steps
This process combines the biotinylated gDNAs from the SUD plate with
query oligos, hybridization reagents, and paramagnetic particles in an
Allele Specific Extension (ASE) plate. The ASE plate is placed in a heat
block and the query oligos for each sequence target of interest are
allowed to anneal to the biotinylated gDNA samples. The gDNA is
simultaneously captured by paramagnetic particles.
Hands-on: ~20 minutes
Incubation: 2–16 hours
Item
Quantity
Storage
Supplied By
OB1 reagent
1 tube per plate
-20ºC
Illumina
OPA reagent
1 tube per plate
-20ºC
Illumina
96-well 0.2 ml skirted
microplate
1 per SUD plate
User
[ ]
Preheat the heat block to 70°C.
[ ]
Turn on the heat sealer to preheat it. Allow 15 minutes.
[ ]
Thaw the OPA reagent tube to room temperature. Vortex the tube, and
then pulse centrifuge to 250 xg. Pour the contents of the OPA tube into a
reagent reservoir.
[ ]
Thaw the OB1 reagent tube to room temperature. Vortex the tube to
completely resuspend the beads. Pour the contents of the OB1 tube into
a second reagent reservoir.Do not centrifuge the OB1 tube.
[ ]
Apply an ASE barcode label to a new microplate.
[ ] 1. Pulse centrifuge the SUD plate to 250 xg.
[ ] 2. Add 10 μl OPA reagent to each well of the ASE plate.
[ ] 3. Add 30 μl OB1 reagent to each well of the ASE plate.
[ ] 4. Carefully remove the heat seal from the SUD plate.
[ ] 5. Transfer 10 μl of biotinylated sample from each well of the SUD plate
(where 10 μl is the entire volume) to the corresponding well of the ASE
plate.
[ ] 6. Using a microplate heat seal, heat-seal the ASE plate (3 seconds).
[ ] 7. Pulse centrifuge the ASE plate to 250 xg.
[ ] 8. Vortex the ASE plate at 1600 rpm for 1 minute or until all beads are
completely resuspended.
[ ] 9. Place the sealed ASE plate on the 70°C heat block and close the lid.
[ ] 10. Immediately reset the temperature to 30°C.
[ ] 11. Allow the ASE plate to cool to 30°C for about 2 hours. The ASE plate
may remain in the heat block for up to 16 hours.
[ ] 12. Proceed to Add MEL.
Catalog # GT-901-1001
Part # 15005074 Rev A
Page 19 of 38
GoldenGate® Genotyping Assay, Manual (Pre-PCR)
Experienced User Card (Universal Array Matrix)
Add MEL
Estimated Time
Consumables
Preparation
Steps
In this process, AM1 and UB1 reagents are added to the ASE plate to
wash away non-specifically hybridized and excess oligos. An enzymatic
extension and ligation master mix (MEL) is added to each DNA sample.
The extension and ligation reaction occurs at 45°C.
Work quickly during the washes to prevent the beads from drying out.
Hands-on: ~30 minutes
Incubation: 15 minutes
Item
Quantity
Storage
Supplied By
AM1 reagent
Bottle
4ºC
Illumina
UB1 reagent
Bottle
4ºC
Illumina
MEL reagent
1 tube per plate
-20ºC
Illumina
[ ]
Remove the ASE plate from the heat block.
[ ]
Preheat the heat block to 45°C for about one hour.
[ ]
Thaw the MEL tube to room temperature.
[ ]
Pour 11 ml AM1 into a reagent reservoir. Add 10 ml for each additional
plate.
[ ]
Pour 11 ml UB1 into a second reagent reservoir. Add 10 ml for each
additional plate.
[ ]
Pour the thawed MEL tube contents into a third reagent reservoir.
[ ] 1. Centrifuge the ASE plate to 250 xg.
[ ] 2. Place the ASE plate on the raised-bar magnetic plate for approximately
2 minutes, or until the beads are completely captured.
[ ] 3. Carefully remove the heat seal from the ASE plate.
[ ] 4. Using an 8-channel pipette with new tips, remove and discard all the
liquid (50 μl) from the wells. Leave the beads in the wells.
[ ] 5. Visually inspect pipette tips after removing solution from each column to
ensure no beads have been removed. If beads are visible in pipette tips,
return the solution to the same wells, allow magnet to re-collect beads,
and change the pipette tips.
[ ] 6. With the ASE plate on the raised-bar magnetic plate, use an 8-channel
pipette with new tips to add 50 μl AM1 to each well of the ASE plate.
[ ] 7. Seal the ASE plate with microplate clear adhesive film.
[ ] 8. Vortex the ASE plate at 1600 rpm for 20 seconds or until all beads are
resuspended.
[ ] 9. Place the ASE plate on the raised-bar magnetic plate for approximately
2 minutes, or until the beads are completely captured.
[ ] 10. Remove the seal from the ASE plate.
[ ] 11. Using the same 8-channel pipette with the same tips, remove all AM1
reagent from each well. Leave the beads in the wells.
Catalog # GT-901-1001
Part # 15005074 Rev A
Page 21 of 38
GoldenGate® Genotyping Assay, Manual (Pre-PCR)
Experienced User Card (Universal Array Matrix)
[ ] 12. Repeat steps 6 through 11 once.
[ ] 13. Remove the ASE plate from the raised-bar magnetic plate.
[ ] 14. Using an 8-channel pipette with new tips, add 50 μl UB1 to each well of
the ASE plate.
[ ] 15. Place the ASE plate onto the raised-bar magnetic plate for approximately
2 minutes, or until the beads are completely captured.
[ ] 16. Using the same 8-channel pipette with the same tips, remove all UB1
reagent from each well. Leave the beads in the wells.
[ ] 17. Repeat steps 13 through 16 once.
[ ] 18. Remove the ASE plate from the raised-bar magnetic plate.
[ ] 19. Using an 8-channel pipette with new tips, add 37 μl MEL to each well of
the ASE plate.
[ ] 20. Seal the plate with microplate clear adhesive film.
[ ] 21. Vortex the plate at 1600–1700 rpm for 1 minute or until the beads are
resuspended.
[ ] 22. Incubate the ASE plate on the preheated 45°C heat block for exactly
15 minutes.
[ ] 23. During the incubation, perform the Make PCR Plate procedure.
[ ] 24. Proceed to Make PCR Plate. Leave the ASE plate at room temperature if
you proceed immediately, or store it at 4ºC for up to 1 hour.
Catalog # GT-901-1001
Part # 15005074 Rev A
Page 22 of 38
GoldenGate® Genotyping Assay, Manual (Pre-PCR)
Experienced User Card (Universal Array Matrix)
Make PCR
Plate
Estimated Time
Consumables
Preparation
Steps
This process adds the Illumina-recommended DNA Polymerase and
optional Uracil DNA Glycosylase (UDG) to the master mix for PCR (MMP
reagent) and creates a 96-well plate for the Inoc PCR process.
Hands-on: ~15 minutes
Item
Quantity
Storage
Supplied By
MMP reagent
1 tube per plate
-20ºC
Illumina
Titanium Taq DNA
Polymerase
64 μl
-20ºC
User
Uracil DNA Glycosylase
(UDG, Optional)
50 μl
-20ºC
User
96-well 0.2 ml skirted
microplate
1 per ASE plate
[ ]
Thaw the MMP tube to room temperature.
[ ]
Apply a PCR barcode label to a new microplate.
User
[ ] 1. Add 64 μl DNA Polymerase to the MMP tube.
[ ] 2. [Optional] Add 50 μl Uracil DNA Glycosylase to the MMP tube.
[ ] 3. Invert the tube several times to mix the contents, and then pour the
contents of the tube into a reagent reservoir.
[ ] 4. Using an 8-channel pipette, add 30 μl of the mixture into each well of the
PCR plate.
[ ] 5. Seal the PCR plate with microplate clear adhesive film.
[ ] 6. Pulse centrifuge to 250 xg, and then place the PCR plate in a lightprotected location.
[ ] 7. Proceed to Inoc PCR Plate.
Catalog # GT-901-1001
Part # 15005074 Rev A
Page 23 of 38
GoldenGate® Genotyping Assay, Manual (Pre-PCR)
Experienced User Card (Universal Array Matrix)
Inoc PCR
Plate
Estimated Time
Consumables
Preparation
Steps
This process uses the template formed in the extension and ligation
process in a PCR reaction. This PCR reaction uses three universal
primers (MMP reagent): two are labeled with fluorescent dyes and the
third is biotinylated. The biotinylated primer allows capture of the PCR
product and elution of the strand containing the fluorescent signal. The
eluted samples are transferred from the ASE plate to the PCR plate.
Hands-on: ~30 minutes
Item
Quantity
Storage
Supplied By
UB1 reagent
Bottle
4ºC
Illumina
IP1 reagent
1 tube per plate
-20ºC
Illumina
[ ]
Remove the ASE plate from the heat block.
[ ]
Reset the heat block to 95°C.
[ ]
Pour 6 ml UB1 into a reagent reservoir.
[ ]
Thaw the IP1 reagent to room temperature. Pour the contents of the
tube into a reagent reservoir.
[ ] 1. Place the ASE plate on the raised-bar magnetic plate for approximately
2 minutes, or until the beads are completely captured.
[ ] 2. Remove the microplate clear adhesive film from the ASE plate.
[ ] 3. Using an 8-channel precision pipette, remove and discard the
supernatant (~50 μl) from all wells of the ASE plate. Leave the beads in
the wells.
[ ] 4. Visually inspect pipette tips after removing solution from each column to
ensure no beads have been removed. If beads are visible in pipette tips,
return the solution to the same wells, allow magnet to re-collect beads,
and change the pipette tips.
[ ] 5. Leaving the plate on the magnet and using an 8-channel precision
pipette with new tips, add 50 μl UB1 to each well of the ASE plate.
[ ] 6. Leave the ASE plate on the raised-bar magnetic plate for approximately
2 minutes, or until the beads are completely captured.
[ ] 7. Remove and discard the supernatant (~50 μl) from all wells of the ASE
plate. Leave the beads in the wells.
[ ] 8. Remove the plate from the magnet.
[ ] 9. Using an 8-channel precision pipette with new tips, add 35 μl IP1 to each
column of the ASE plate.
[ ] 10. Seal the plate with microplate clear adhesive film.
[ ] 11. Vortex at 1800 rpm for 1 minute, or until all the beads are resuspended.
[ ] 12. Place the plate on the 95°C heat block for 1 minute.
[ ] 13. Place the ASE plate back onto the raised-bar magnetic plate for
2 minutes or until the beads have been completely captured.
Catalog # GT-901-1001
Part # 15005074 Rev A
Page 25 of 38
GoldenGate® Genotyping Assay, Manual (Pre-PCR)
Experienced User Card (Universal Array Matrix)
[ ] 14. Using an 8-channel pipette with new tips, transfer 30 μl supernatant from
each well in the first column of the ASE plate to the first column of the
PCR plate.
[ ] 15. Repeat for each column of the ASE plate. Change tips between column
dispenses.
[ ] 16. Seal the PCR plate with the appropriate PCR plate-sealing film for your
thermocycler.
[ ] 17. Immediately transfer the PCR plate to the thermocycler.
[ ] 18. Proceed to Cycle PCR.
This concludes the Pre-PCR processes for the manual GoldenGate Genotyping
Assay for VeraCode. If you remove materials such as experienced user cards from
the Pre-PCR lab, do not return with them into the Pre-PCR lab at any time.
Catalog # GT-901-1001
Part # 15005074 Rev A
Page 26 of 38
GoldenGate® Genotyping Assay, Manual (Post-PCR)
Experienced User Card (Universal Array Matrix)
Cycle PCR
Estimated Time
Steps
This process thermal cycles the PCR plate to fluorescently label and
amplify the templates generated in the pre-PCR process.
Thermal Cycle: ~2 hours 45 minutes
[ ] 1. Place the sealed plate into the thermocycler and run the thermocycler
program shown in this table.
Table 1
Thermocycler Program
X 34
Temperature
Time
37°C
10 minutes
95°C
3 minutes
95°C
35 seconds
56°C
35 seconds
72°C
2 minutes
72°C
10 minutes
4°C
5 minutes
[ ] 2. Do one of the following:
Catalog # GT-901-1001
Part # 15005074 Rev A
•
Proceed immediately to Bind PCR. Store the PCR plate at room
temperature in a light-protected drawer.
•
Seal and store the PCR plate at -20°C overnight.
Page 27 of 38
GoldenGate® Genotyping Assay, Manual (Post-PCR)
Experienced User Card (Universal Array Matrix)
Bind PCR
Estimated Time
Consumables
Preparation
Steps
In this process, MPB reagent is added to the PCR plate and the solution
is transferred to a filter plate. The filter plate is incubated at room
temperature to bind the biotinylated strand to paramagnetic particles,
thus immobilizing the double-stranded PCR products.
Hands-on: ~30 minutes
Incubation: 1 hour
Item
Quantity
Storage
Supplied By
MPB reagent
1 tube per PCR plate 4ºC
Illumina
0.45 μM clear Styrene filter
plate with lid
1 per PCR plate
User
[ ]
Vortex the MPB tube several times or until the beads are well
resuspended. Pour the contents of the tube into a non-sterile reagent
reservoir.
[ ]
Write the PCR plate barcode number in the space provided on a “Filter
Plate: GS ____________-PCR” label. Apply the label to the top surface of
the filter plate, adjacent to column 12.
[ ] 1. Pulse centrifuge the PCR plate to 250 xg.
[ ] 2. Place new tips onto an 8-channel pipette.
[ ] 3. Add 20 μl resuspended MPB into each well of the PCR plate.
[ ] 4. Set an 8-channel pipette to 85 μl to allow space for bubbles, and attach
new tips.
[ ] 5. Pipette the solution in the PCR plate up and down several times to mix
the beads with the PCR product.
[ ] 6. Transfer the mixed solution into the first column of the filter plate. There
should be about 70 μl fluid in each well.
[ ] 7. Repeat step 6 for each column of the PCR plate. Change tips between
column dispenses.
[ ] 8. Cover the filter plate with its lid and store it at room temperature,
protected from light, for 60 minutes.
[ ] 9. Proceed to Make HYB Plate.
Catalog # GT-901-1001
Part # 15005074 Rev A
Page 29 of 38
GoldenGate® Genotyping Assay, Manual (Post-PCR)
Experienced User Card (Universal Array Matrix)
Make HYB
Plate
Estimated Time
Consumables
Preparation
Steps
In this step, the single-stranded, fluor-labeled PCR product from the Filter
plate is washed, eluted into the INT plate, then transferred to a new plate
(HYB plate) suitable for mating to the Universal Array Matrices (UAMs).
Hands-on: ~45 minutes
Item
Quantity
Storage
Supplied By
0.1N NaOH
Bottle
2°C to 8°C
User
UB2 reagent
Bottle
Room
temperature
Illumina
MH1 reagent
1 tube per INT plate Room
temperature
Illumina
[ ]
Apply an INT barcode label to a new 96-well V-bottom plate.
[ ]
Apply a HYB barcode label to a Cliniplate 384-well microplate.
[ ]
Pour 10 ml UB2 into a sterile trough.
[ ]
Pour 5 ml 0.1N NaOH into another sterile trough.
[ ]
Pour the contents of one MH1 tube into another sterile trough.
Make HYB
[ ] 1. Place the Filter plate adapter on an empty 96-well V-bottom plate (Waste
plate).
[ ] 2. Place the Filter plate containing the bound PCR products onto the Filter
plate adapter.
[ ] 3. Centrifuge at 1000 xg for 5 minutes at 25°C.
[ ] 4. Remove the Filter plate lid.
[ ] 5. Using an 8-channel pipette (with new tips), add 50 μl UB2 from the sterile
trough to each column of the Filter plate.
[ ] 6. Re-lid the Filter plate.
[ ] 7. Centrifuge at 1000 xg for 5 minutes at 25°C.
[ ] 8. Place new tips onto an 8-channel pipette.
[ ] 9. Dispense 30 μl MH1 from the trough to all columns of the INT plate.
[ ] 10. Replace the Waste plate with the INT plate, and discard the Waste plate.
[ ] 11. Orient the INT plate such that well A1 of the Filter plate matches well A1
of the INT plate.
[ ] 12. Place new tips onto the 8-channel pipette.
[ ] 13. Dispense 30 μl 0.1N NaOH to all wells of the Filter plate.
[ ] 14. Re-lid the Filter plate.
[ ] 15. Centrifuge immediately at 1000 xg for 5 minutes at 25°C.
[ ] 16. Discard the Filter plate; save the adapter.
[ ] 17. Gently mix the contents of the INT plate by moving it from side to side,
without splashing.
Catalog # GT-901-1001
Part # 15005074 Rev A
Page 31 of 38
GoldenGate® Genotyping Assay, Manual (Post-PCR)
Experienced User Card (Universal Array Matrix)
[ ] 18. Place new tips onto the 8-channel pipette.
[ ] 19. Using an 8-channel precision pipette, transfer 30 μl UB2 from its sterile
trough into the first marked column of the HYB plate.
[ ] 20. Repeat steps 18 and 19 for each column of the HYB plate.
[ ] 21. Place new tips onto an 8-channel pipette.
[ ] 22. Slowly pipette up and down several times to mix contents in the INT
plate, then transfer 50 μl neutralized hyb solution from the first column of
the INT plate into the first marked column of the HYB plate.
NOTE
Dispense sample A1 from the INT plate into well A1 of the
HYB plate. Dispense sample B1 from the INT plate into well
C1 of the HYB plate, etc. Eject and discard pipette tips.
[ ] 23. Repeat steps 21 and 22 for all columns of the INT plate.
[ ] 24. Using microplate clear adhesive sealing film, seal the plate.
[ ] 25. Centrifuge at 3000 xg for 4 minutes at 25°C.
[ ] 26. Do one of the following:
Catalog # GT-901-1001
Part # 15005074 Rev A
•
If the HYB plate is not used immediately, store it at -20°C.
•
Proceed to Hyb to UAM.
Page 32 of 38
GoldenGate® Genotyping Assay, Manual (Post-PCR)
Experienced User Card (Universal Array Matrix)
Hyb to UAM
Estimated Time
Consumables
Preparation
Steps
Once the HYB plate has been assembled, the samples are ready for
hybridization. The UAM is prepared for hybridization through a series of
washes that clean and rehydrate the beads on the fiberoptic bundles.
Next, the HYB plate is paired with the UAM and hybridized overnight,
using the natural cooling of the oven from 60°C to 45°C to anneal the
fluor-labelled PCR products to the complementary sequences on the array.
The plate is washed on the following day. Fill in the lab tracking form as
you work.
Hands-on: 30 minutes
Incubation: 30 minutes, then 14 hours
Item
Quantity
Storage
Supplied By
UB2 reagent
Bottle
Room
temperature
Illumina
0.1N NaOH
Bottle
4ºC
User
Universal Array Matrix and
accompanying CD
1 per HYB plate
Illumina
[ ]
Preheat the Illumina Hybridization Oven to 60°C and allow the
temperature to stabilize. This takes about 45 minutes.
[ ]
If the oven contains a rocker platform, remove it. Position the oven rack
off the floor so that the heating element vents do not direct highertemperature air directly towards the arrays.
[ ]
If the HYB plate has been frozen, thaw it completely at room temperature
in a light-protected drawer.
[ ]
Label an OmniTray “UB2” and dispense 70 ml UB2 into the tray.
[ ]
Label another OmniTray “NaOH” and dispense 60 ml 0.1N NaOH into
the tray.
Wash the UAM
[ ] 1. Carefully place the UAM, with the barcode facing up and the fiber
bundles facing down, into the UB2 conditioning tray.
[ ] 2. Agitate the UAM gently for 10 seconds only, moving it up and down to
remove bubbles from the bottoms of the fiber bundle arrays.
[ ] 3. After 3 minutes, move the UAM into the 0.1N NaOH tray.
[ ] 4. After 30 seconds, move the UAM back into the UB2 tray.
[ ] 5. Allow the UAM to sit in the UB2 tray for at least 30 seconds to neutralize
the NaOH. The UAM may sit in the UB2 conditioning tray for up to
2 hours.
Assemble the UAM Hyb Cartridge
[ ] 1. Centrifuge the sealed HYB plate to 3000 xg for 4 minutes.
[ ] 2. Carefully remove the clear adhesive film from the HYB plate.
Catalog # GT-901-1001
Part # 15005074 Rev A
Page 33 of 38
GoldenGate® Genotyping Assay, Manual (Post-PCR)
Experienced User Card (Universal Array Matrix)
[ ] 3. Slide the HYB plate into the UAM Hyb Cartridge, using the metal tabs as
guides and matching the notched corner of the plate with the notched
corner of the frame.
[ ] 4. Using both hands, place the UAM into the UAM Hyb Cartridge from
above, using the using the metal pegs as guides. Slowly lower the UAM
until it is firmly seated on top of the HYB plate.
Incubate UAM / HYB Plate Pair
[ ] 1. Place the HYB plate/UAM pair(s) into the preheated 60ºC hybridization
oven. You can hybridize up to four pairs at one time.
[ ] 2. After 30 minutes, reset the oven to 45°C.
[ ] 3. Incubate for at least 14 hours.
[ ] 4. After the incubation proceed to Wash UAM.
Catalog # GT-901-1001
Part # 15005074 Rev A
Page 34 of 38
GoldenGate® Genotyping Assay, Manual (Post-PCR)
Experienced User Card (Universal Array Matrix)
Wash UAM
Estimated Time
Consumables
Preparation
Steps
In this process, the hybridized UAMs are washed in IS1 and UB2,
preparatory to imaging. This part of the assay protocol occurs on the
same day that you image the UAM. Fill in the lab tracking form as you
work.
Hands-on: 30 minutes
Dry: 20 minutes
Item
Quantity
Storage
Supplied By
IS1 reagent
Bottle
Room
temperature
Illumina
UB2
Bottle
Room
temperature
Illumina
95% EtOH
Bottle
Room
temperature
User
Sec-butanol
Bottle
Room
temperature
User
OmniTrays
4 per UAM
User
[ ]
Turn on the BeadArray™ Reader at least one hour before scanning.
[ ]
Label two OmniTrays “UB2,” and dispense 70 ml UB2 into each tray.
[ ]
Label a third tray “IS1” but do not fill it yet.
[ ]
The fourth tray will be used to dry the array.
Cover each tray with its plastic cover until ready for use.
Make IS1 Mixture
[ ] 1. Make 940 ml of a 50/50 mixture of 95% EtOH and sec-butanol.
[ ] 2. Remove the cap from a new IS1 bottle and add the mixture to it.
[ ] 3. Replace the cap on the IS1 bottle. Shake to mix well.
[ ] 4. Add 70 ml of the IS1 mixture to the tray marked “IS1.”
Wash SAM
[ ] 1. Remove the UAM to be imaged from the hybridization oven.
[ ] 2. Separate the UAM from the HYB plate.
[ ] 3. Seal the HYB plate with microplate clear adhesive film and store it at
-20°C.
[ ] 4. Place the array immediately into the first UB2 tray. Gently agitate the
array by shaking it side to side 10 times. Leave in the UB2 tray for
1 minute at room temperature.
[ ] 5. Transfer the array to the second UB2 tray. Gently agitate the array by
shaking it side to side 10 times. Leave in the UB2 tray for 1 minute at
room temperature.
Catalog # GT-901-1001
Part # 15005074 Rev A
Page 35 of 38
GoldenGate® Genotyping Assay, Manual (Post-PCR)
Experienced User Card (Universal Array Matrix)
[ ] 6. Transfer the array into the IS1 tray and set the timer for 5 minutes. Lift the
UAM out of the IS1 several times.
[ ] 7. Remove the array from the IS1 tray and place it on an empty OmniTray,
fiber bundles up.
[ ] 8. Air-dry for 20 minutes in a closed drawer at room temperature.
[ ] 9. Proceed to Image UAM.
Catalog # GT-901-1001
Part # 15005074 Rev A
Page 36 of 38
GoldenGate® Genotyping Assay, Manual (Post-PCR)
Experienced User Card (Universal Array Matrix)
Image UAM
Estimated Time
Preparation
Steps
In this process, the UAMs are imaged in the Illumina BeadArray Reader.
The BeadArray Reader uses lasers to excite the Cy3 and Cy5 fluors of
the single-stranded PCR products bound to the beads of the fiberoptic
bundles. Light emissions from these fluors are separately recorded in
high-resolution images of the fiber optic bundles. Fill in the lab tracking
form as you work.
Scanning: 1.5 hours
[ ]
Turn on the BeadArray Reader at least one or two hours before
beginning a scan, to allow the lasers to stabilize.
First Use of the BeadArray Reader Today
[ ] 1. Turn the power switch to the ON position.
[ ] 2. Wait for the Ready indicator to stop flashing.
[ ] 3. Double-click the BeadScan icon on the BeadArray Reader PC desktop.
[ ] 4. Enter your user name, and then click Scan.
Set Up the Scan
[ ] 1. From the BeadScan Docking Fixture list box, select Array Matrix.
[ ] 2. If you want to change any scan settings, click Edit.
[ ] 3. Change any of the settings and do one of the following:
•
Click Save for This Scan to apply the changed settings to the current
scan only.
•
Click Save for All Scans to apply the changed settings to this and all
future scans.
[ ] 4. Just before imaging, use canned air to clean the proximal ends (barcode
side) of the UAM fiberoptic bundles.
[ ] 5. Place the UAM into the BeadArray Reader tray.
[ ] 6. Scan the UAM barcode with the hand-held barcode scanner.
The barcode appears in the Barcode box in the BeadScan software. The
tray closes automatically.
[ ] 7. If you do not want to scan one or more sections (fiber array bundles) on
the SAM, click the sections in the UAM image to deselect them.
[ ] 8. Click the Browse (...) button beside the Scan Settings box to open the
Select Scan Setting dialog box.
[ ] 9. Double-click GoldenGate Genotyping to apply the correct settings to
the scan.
[ ] 10. Click Scan.
Catalog # GT-901-1001
Part # 15005074 Rev A
Page 37 of 38
Was this manual useful for you? yes no
Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Download PDF

advertisement