Affymetrix HG-U133 Set Gene Chip Protocol List
Samples:
Evening before:
1. Check OD of total RNA and run a final gel.
2. Calculate how much RNA is needed for (8ug) for labeling.
Add that amount to a new tube and speedvac on high until
dry.
3. Resuspend RNA in 7.5 ul water and keep at -20C O/N.
4. Heat RNA at 37C for 5 minutes.
5. Vortex and spin briefly.
Day 1:_______________________
cDNA Synthesis
First
1. Make master mix of water and T7 Primer:
Master Mix ___x
DEPC-treated water (Ambion)
3 ___
T7 Primer (Affymetrix)
1 ___
Total
4ul
2. Add 4ul Master
Mix to each pcr tube, then add 7 ul RNA, mix by pipetting.
3. Incubate at 70C for 10 minutes, spin, put on ice.
4. Make master mix:
Master Mix ___x
Keep on ICE:
5X First
0.1 M DTT (Invitrogen)............................2 ___
10mM dNTP Mix (Invitrogen)
...1 ___
SSII RT (Invitrogen)
...2 ___
Total
9ul
5. Add 9ul Master
Mix to each pcr tube, mix by pipetting.
6. Incubate at 42C for 1.5 hrs, put on ice, centrifuge.
Start:____________ End:___________
Second
7. Make master mix:
Master
Mix ___x
DEPC- treated water (Ambion)
91 ___
5X Second
10mM dNTP mix (Invitrogen)
.....3 ___
10 U/ul DNA ligase (New England Biolabs)
1 ___
10 U/uL E.Coli DNA Polymerase I
.4 ___
(
2 U/uL Rnase H (New England Biolabs)
..0.5 ___
Total 130 ul
8. Add 130 uL to each tube, mix by pipeting, centrifuge,
incubate at 16C for 2 hrs.
Start:____________ End:___________
9. Spin down. Add 2 uL [10U] T4 DNA polymerase (New England Biolabs), mix by pipeting.
10. Incubate at 16C for 15 minutes.
11. Add 10 uL 0.5M EDTA.
Cleanup Part I (Do in hood)
1. Spin PLG (phase lock gel tube) at full speed for 2
minutes.
2. Add 162 uL of Phenol:Chloroform:isoamyl alcohol (Invitrogen) to new 1.5 mL tube.
(use lower, clear organic layer)
3. Add sample (162 ul) to Phenol/Chloroform tube, vortex.
4. Transfer all liquid to PLG tube, DO NOT VORTEX!
5. Spin at full speed for 2 minutes.
6. To a new 1.5 ml tube, make a master mix:
Master
Mix ___x
NH4OAc (Sigma-Aldrich)
90 ___
Glycogen (Ambion)
4 ___
Total
94
7. Transfer upper aqueous phase of PLG tube to NH4OAc/Gly
tube, finger vortex.
*********be sure to mix everything well before adding ethanol!***************
8. Add 600 uL absolute, -20C ethanol
9. Finger vortex and store in -20C overnight.
Day 2:_______________________
Cleanup Part II
1. Take samples out of freezer and
let sit for 15 minutes on ice.
2. Spin 20 minutes at full speed in
RT.
3. Add 500 uL 75%, -20C ethanol,
vortex, spin at full speed 10 minutes.
4. Pipet out supernatant.
5. Add 500 uL 75%, -20C ethanol,
vortex, spin at full speed 10 minutes.
6. Pipet out supernatant and use
speed vac to dry pellet.
7. Dissolve WELL in 22 uL DEPC-treated
water, spin, incubate at 37C for 5 min and spin again.
IVT Reaction Setup
1. Make
master mix (Enzo
BioArray HighYield RNA Transcript Labeling Kit, Affymetrix):
Master
Mix ___x
Solution 1
2 ___
Solution 2
2 ___
Solution 3
2 ___
Solution 4
2 ___
Solution 5
1 ___
2. Setup
and mix by pipetting.
Master mix
..9
cDNA
...11
Total
.20
3. Use
thermalcycler program set at 37C (~12 hrs): labeling
Day 3:_______________________
IVT Reaction Cleanup
(Qiagen
RNeasy Mini Kit)
1. Take
samples out of thermalcycler, RT.
2. Label
eppendorfs, add 350 ul Buffer RLT.
3. Add 80
ul water to IVT sample tube, mix well, add to RLT tube, mix well then pipet
from RLT tube to sample tube, mix well again, and then pipet all remaining
liquid from sample tube to RLT tube, vortex- PUT RLT away!
4. Add 250
ul 100% ethanol to RLT tubes, mix, then transfer all liquid to RNeasy column.
5. Spin at
8,000 rpm for 1 minute.
6. Dump
flow through, add 500 ul Buffer RPE,
spin at 8,000 rpm for 1 minute.
7. Discard
collection tube, put Rneasy column in new 2 ml collection tube.
8. Spin at
full speed for 2 minutes.
9. Discard
collection tube, put column in new 1.5 ml collection tube.
10. Add 55
ul DEPC-treated water, spin at 10,000 rpm for 2 minutes.
11. Measure
OD (use 1x TE).
Fragmentation and Hybridization
1. 1 hour
prior: Turn heat bath on to 99C.
2. Take OD
and calculate uL needed for 15 ug cRNA.
3. Add that
amount to a new tube and dry in speedvac.
4.
Resuspend in 32 uL water, incubate 5 minutes at 37C.
5. Take
chips out of cold room and place on bench unwrapped with window side down.
6. In pcr
tube add and mix by pipeting:
5x fragmentation buffer (GeneChip sample cleanup module)
..8
cRNA
............................32
7. Run
thermalcycler program: frag_94 for
35 minutes.
8. Make
master mix (keep on ice):
(5x is max per tube)
Master
mix ___x ___x ___x
DEPC-treated water
.. 97 ___ ___ ___
2x
Hybridization buffer
..150 ___ ___ ___
Acetylated
BSA (Invitrogen)
3 ___ ___ ___
Herring
Sperm DNA (Promega)
3 ___ ___ ___
Oligo B2 (Affymetrix)
7 ___ ___ ___
Total
260 ul
9. Make 300
uL 1X hybridization buffer per chip (from 2X hyb buffer).
10. Label
chips on both sides and pierce top septa with pipet tip.
11. Turn on
hybridization oven to 45C.
12. Add 260
uL master mix to each eppendorf tube. Add 40 uL of fragmented cRNA to tube with
master mix. Vortex.
13. Pierce
top of tube with syringe.
14. Put
tubes in 99C heat block (filled with water) for 5 minutes.
15. In the
meantime make sure the chips are at RT then add 300 uL 1X hyb to each chip and
shake the chip slowly to distribute buffer on chip. Leave for about 10 minutes.
16. Spin
tubes at full speed for 5 minutes.
17. Remove
1X hyb from chips.
18. Add
sample to chip, leaving a small air bubble.
19. Put in
hyb over for 16 hours.
Start:____________ End:___________
Day 4:_______________________
1. Take chips out of hyb oven.
2. Put pipet tip in top septum to vent and remove hyb
cocktail and put in separate labeled eppendorf tube for storage.
3. Add 300 uL
4. Prime fludics station with
5. Make master mix for 1st and 3rd
stain:
Master
mix 2x
DEPC-treated
water
540
2X stain
buffer
..600
BSA (Invitrogen)
48
Phycoerythrin
Streptavidin (Molecular
Probes)
..12
6. Vortex and take out 600 uL and put into another tube.
7. Make 2nd stain:
DEPC-treated
water
266
2X stain
buffer
..300
BSA (Invitrogen)
24
Goat IgG (Sigma-Aldrich)
...6
Biotinylated
antibody (Vector
Laboratories)
...3.6
8. Vortex and take all three stains to the fluidics station.
9. Set programs on fluidics station and run the EukGEs2v4
protocol.
10. Load chip and 1st stain and start protocol.
1st
stain: 42 minutes
2nd
stain: 10 minutes
3rd
stain: 27 minutes
11. Check chips for bubbles before scanning. If there are
bubbles, empty liquid, wait a few minutes and add more
12. Place white round stickers on top of vents to prevent
leakage into scanner.
13. On new scanner enter expt info and start scan.