TruSeq HT Workflow
Adapted from Illumina TruSeq Sample Preparation Guide (version July 2012)
Modified for:
-
plate based on-bead cleanups using AmPureXP
-
double size selection using beads (0.5x > 0.75x)
BEFORE STARTING
Make sure you have ready:
-
AMPureXP beads
-
PEG solution (20% PEG 6000(or20000), NaCl
-
Ethanol
-
30C oven
-
37C oven
-
96-well plate magnet
-
2x round bottom 96-well plates (test that they are compatible with
magnet for AMPureXP cleans)
-
1x 96-well PCR plate
-
Microseal B plate seals
PROTOCOL
DNA and shearing
DNA
-
500-1000ng good quality DNA (high molecular weight)
-
quantified by Qubit
Shearing DNA using Bioruptor
-
requires optimization to determine time, intensity and cycles
-
these will change depending on DNA source (genomic, PCR amplicon) and
concentration
-
also concentration dependent
-
check smear by running gel
-Current exp (Long range PCR amplicons)
-
DNA: 500ng in 50ul – standard mcrocentrifuge tubes
-
Power: High
-
Cycles: 30sec ON / 30 sec off
-
Time: 22 mins (11mins > pulse spin > 11 mins)
Perform End repair (starting volume 50 ul in plate)
1.
Before starting
a. remove End Repair Mix and thaw at room temperature
b. AMPureXP beads: bring to room temp for 30 mins
c. make sure 30C oven is on
d. thaw resuspension buffer
2.
Add 10 ul Resuspension buffer to each sample well
a. Use 8-well strips with 120 ul in each well followed by multichannel to
dispense
3.
4.
Add 40 ul of End Repair Mix to each sample well
a.
Seal plate with Microseal B adhesive
b.
Vortex 30 secs Shake plate at 1,800 RPM for 2 mins
c.
Centrifuge plate at 280 g for 1 min
Incubate plate at 30C for 30 mins, spin @ 280 g for 1 min, after which
remove seal
5.
AMPureXP clean (1x ratio @ 100ul starting volume)
a.
b.
vortex beads and make fresh 80% ethanol (need 400 ul per well)
i.
96 wells = 400 ul x 96 = 38,400 ul so make 40 mls
ii.
32 mls EtOH + 8 mls water
create batch of diluted bead mix by combining 20 ul AMPure beads
with 80 ul PEG20 solution (don’t need more than 20 ul beads
i.
c.
For 96 samples mix 1920 ul beads with 7680 ul PEG20
Add 100 ul of bead/PEG20 mix to each well
i.
Pipette up/down 20X to mix
ii.
Seal plate with Microseal B adhesive
iii.
Shake plate at 1,800 RPM for 2 mins
iv.
incubate for 10 mins
d.
Place plate on magnet for 5 mins (make sure liquid appears clear)
e.
While still on the magnet, use aspirator pump to remove 200 ul and
discard (careful not to touch the beads) –
f.
Add 200 ul 80% EtOH to each well, wait 30 secs, and then remove
using aspirator pump
g.
Repeat last step once for total of two washes
h.
Allow plate to air dry for 5-10 mins (or until no EtOH can be detected)
i.
Remove plate from magnet, and resuspend beads in 17.5 ul
Resuspension Buffer
j.
Add 17.5 ul H20 with 12.5 ul A-tailing buffer (next step), and
resuspend beads by pipetting up and down
k.
Proceed straight to next step
*** SAFE STOPPING POINT (seal plate and store in -20C freezer up to 7 days)
Adenylate 3’ Ends (starting volume: 17.5 ul in plate)
1.
Before starting
a. thaw the A-tailing mix at room temperature
b. if sample plate was in freezer, thaw at RT, and spin at 280 g for 1 min
c. Preheat oven to 37C
2.
Add 12.5 ul of A-tailing mix to each well (if already added from previous
step)
a. Seal plate with Microseal B adhesive
b. Vortex for 30 secs
c. Centrifuge plate at 280 g for 1 min
3.
Incubate plate at 37C for 30 mins
Ligate Adapters (starting volume: 30 ul in plate)
1.
Before starting
a. thaw at room temperature
i. DAP plate
1. ~10 mins, after which visually check to make sure fully
thawed
2. remove adapter plate tape seal
3. Centrifuge plate at 280 g for 1 mins
4. Remove plastic cover (save if not using entire plate
now)
ii. Stop Ligation buffer
b. make sure 30C oven is on
2. Immediately before use, remove Ligation Mix from freezer
3. Add 2.5 ul Resuspension buffer to each well
4. Add 2.5 ul Ligation Mix to each well (return Ligation Mix to freezer
immediately after use)
5. Pierce holes in DAP plate using either a clean, semi skirted 96-well plate, or
clean 8-well strip (new strip each time – DO NOT CROSS CONTAMINATE)
6. Using 8-well multichannel pipette, transfer 2.5 ul of adapter from DAP plate
to corresponding well in sample plate
a. Seal plate with Microseal B adhesive
b. Vortex for 30 secs Shake plate at 1,800 RPM for 2 mins
c. Centrifuge plate at 280 g for 1 mins
7. Incubate sample plate at 30C for 10 mins
8. Remove adhesive seal
9. Add 5 ul of Stop Ligation Buffer to each sample well
a. Seal plate with Microseal B adhesive
b. Vortex for 30 secs Shake plate at 1,800 RPM for 2 mins
c. Centrifuge plate at 280 g for 1 mins
AMPureXP Clean and bead double size selection (0.55X + 0.75X size
selection; starting volume: 42.5 ul in plate)
1. Before starting
a. AMPureXP beads: bring to room temp for 30 mins
b. make 80% EtOH (if 96 samples, make 40 mls)
2. Add 57.5 ul resuspension buffer to each well
3. Add 55 ul PEG20 to each well
a. Pipette up/down to mix
b. Seal plate with Microseal B adhesive
c. Shake plate at 1,800 RPM for 2 mins
d. incubate for 10 mins
4. Place plate on magnet for 5 mins (make sure liquid appears clear)
5. KEEPING THE SUPERNATANT, remove supernatant to new plate (150 ul)
6. Add 20 ul of AMPureXP beads to each sample well in new plate
a. Pipette up/down to mix
b. Seal plate with Microseal B adhesive
c. Shake plate at 1,800 RPM for 2 mins
d. incubate for 10 mins
7. Place plate on magnet for 5 mins (make sure liquid appears clear)
8. While still on the magnet, use aspirator pump to remove 200 ul and discard
(careful not to touch the beads)
9. Add 200 ul 80% EtOH to each well, wait 30 secs, and then remove using
aspirator pump
10. Repeat last step once for total of two washes
11. Allow plate to air dry for 10 mins (or until no EtOH can be detected)
12. Remove plate from magnet, and resuspend beads in 22.5 ul Resuspension
Buffer, and wait 10 mins
13. Place plate back on magnet for 5 mins
14. Transfer 20 ul of samples to new PCR plate
*** SAFE STOPPING POINT (seal plate and store in -20C freezer up to 7 days)
Enrich DNA Fragments (starting volume: 20 ul in plate)
1. Before starting
a. Thaw PCR Master Mix and PCR Primer Cocktail at room temperature.
Pulse spin and keep on ice once thawed.
b. Program PCR machine
i. 98C for 30 seconds
ii. 10 cycles of
1. 98C for 10 seconds
2. 60C for 30 seconds
3. 72C for 30 seconds
iii. 72C for 5 mins
iv. hold at 10C
2. add 5 ul of PCR Primer Cocktail to each sample well
3. add 25 ul of PCR Master Mix to each sample well
a. Seal plate with Microseal B adhesive
b. Vortex to mix
c. Centrifuge plate at 280 g for 1 mins
4. Proceed with PCR
Clean Up PCR (starting volume: 50 ul in plate)
5. Once PCR is finished, transfer PCR reactions back into previous plate used for
second step of size selection and clean with AMPureXP beads (already in
plate)
6. Add 37.5 ul PEG20 to each well in plate (0.75X)
i. Pipette up/down 20X to mix
ii. Seal plate with Microseal B adhesive
iii. Shake plate at 1,800 RPM for 2 mins
iv. incubate for 10 mins
7. Place plate on magnet for 5 mins (make sure liquid appears clear)
8. While still on the magnet, remove 200 ul and discard (careful not to touch the
beads)
9. Add 200 ul 80% EtOH to each well, wait 30 secs, and then remove
10. Repeat last step once for total of two washes
11. Allow plate to air dry for 10 mins (or until no EtOH can be detected)
12. Remove plate from magnet, and resuspend beads in 35 ul Resuspension
Buffer
i. Pipette up/down 20X to mix
ii. Seal plate with Microseal B adhesive
iii. Shake plate at 1,800 RPM for 2 mins
iv. incubate for 10 mins
13. Place plate on magnet for 5 mins (make sure liquid appears clear)
14. Transfer entire volume of each well into a new plate
*** SAFE STOPPING POINT (seal plate and store in -20C freezer up to 7 days)