PROTOCOLS
GS-FLX Pyrosequencing Protocols
Overview Genome Sequencer Guides and Manuals
GX FLX Shotgun DNA Library Preparation QuickGuide
GS FLX Short Paired End DNA Library Preparation QuickGuide
GS FLX Long Paired End DNA Library Preparation QuickGuide
GS FLX Amplicon DNA Library Preparation QuickGuide
GS FLX emPCR QuickGuide (small preparation)
GS FLX emPCR QuickGuide (large preparation)
GS FLX emPCR Sequencing QuickGuide

Genomic DNA labeling and Hybridization The protocol is adopted from Patrick Brown's method developed for microarray-based comparative genomic hybridization (this can be found here). Genomic DNA can be labeled with a simple random-priming protocol based on Gibco/BRL's Bioprime DNA Labeling kit, though nick translation protocols work too. I routinely use the BioPrime labeling kit (Gibco/BRL) as a convenient and inexpensive source of random octamers, reaction buffer, and high concentration klenow (do not use the dNTP mix provided in the kit), though other sources of random primers and high concentration klenow work as well.

1. Add 2 ug DNA of the sample to be labeled to an eppindorf tube. Note: For high complexity DNAs (e.g. human genomic DNA), the labeling reaction works more efficiently if the fragment size of the DNA is first reduced. I routinely accomplish this by restriction enzyme digestion (usually DpnII, though other 4-cutters work as well). After digestion, the DNA should be cleaned up by phenol/chloroform extraction / EtOH precipitation (Qiagen PCR purification kit also works well).
2. Add ddH₂0 or TE 8.0 to bring the total volume to 21 ul. Then add 20 ul of 2.5X random primer / reaction buffer mix. Boil 5 min, then place on ice. 2.5X random primer / reaction buffer mix:
   125 mM Tris 6.8
   12.5 mM MgCl₂
   25 mM 2-mercaptoethanol
   750 ug/ml random octamers
3. On ice, add 5 ul 10X dNTP mix. 10X dNTP mix:
   1.2 mM each dATP, dGTP, and dTTP
   0.6 mM dCTP
   10 mM Tris 8.0, 1mM EDTA
4. Add 3 ul Cy5-dCTP or Cy3-dCTP (Amersham, 1 mM stocks) Note: Cy-dCTP and Cy-dUTP work equally well. If using Cy-dUTP, adjust 10X dNTP mix accordingly.
5. Add 1 ul Klenow Fragment. Note: High concentration klenow (40-50 units/ul), available through NEB or Gibco/BRL (as part of the BioPrime labeling kit), produces better labeling.
6. Incubate 37 degrees C for 1 to 2 hours, then stop reaction by adding 5 ul 0.5 M EDTA pH 8.0.

Purification of the Probe using QIAquick PCR purification kit. (modified in our facility)

1. Combine Cy3 and Cy5 reactions.
2. Add 500 ul Buffer PB.
3. Apply to QIAquick column. Spin at 13000 rpm for 1 min.
4. Re-apply flow-through for optional binding. Spin at 13000 rpm for 1 min.
5. Decant flow-through.
6. Add 750 ul Buffer PE. Spin at 13000 rpm for 1 min. Decant flow-through.
7. Spin at maximum speed for 1 min to dry column.
8. Transfer column into the new 1.5ml tube.
9. Add 23 ul of ArrayHyb low temp hybridization buffer (Sigma) to the center of the membrane.
10. Spin at 13000 rpm for 1 min.

Array Hybridization

1. Pre-warm hybridization chamber at 70^oC on Slide Moat (Boekel Scientific).
2. To the eluted probe 3 ul of salmon sperm DNA (1mg/ml stock) and 1.5 ul of tRNA(9.2mg/ml) to block non-specific hybridization.
3. Denature the probe at 65^oC for 5 min. (due to low temp ArrayHyb buffer).
4. Centrifuge at 13000 rpm for 10-20 min to pellet any particulates.
5. Pipet 10-15 ul of water into the wells or gaps located on either end of the hybridization chamber to prevent drying of array during hybridization.
6. Place probe on the center of a cover slip. Cover the probe carefully with the array.
7. Quickly transfer the slide into the hybridization chamber. Tighten the chamber.
8. Incubate the hybridization chamber in 50^oC water bath for overnight.

RNA labeling and Hybridization Total bacterial RNA Reverse transcription reaction

1. Prepare reverse transcription reactions. One reaction for sample RNA and one for reference RNA.

Ingredients	Cy3 rxn	Cy5 rxn
Total RNA	x ul	y ul
Random hexamer(1 ug/ul)	1 ul	1 ul
Nuclease-free water	to 14 ul	to 14 ul

• Incubate at 65oC for 10 min.
• Cool on ice.
• Add followings.

Ingredients	Cy3 rxn	Cy5 rxn
5X 1st strand buffer	6 ul	6 ul
0.1 M DTT	3 ul	3 ul
dNTP mix(low dCTP)**	0.6 ul	0.6 ul
SUPERase-In (20 ug/ul) (Ambion Inc.)	1.5 ul	1.5 ul
Cy-dCTP dye (1mM/ul)(Amersham)	3 ul Cy3-dCTP	3ul Cy5-dCTP

** dNTP mix: 25 mM of each dATP, dGTP, and dTTP, 10 mM of dCTP

1. Incubate at 42^oC fro 2 min.
2. Add 2 ul of Superscript II reverse transcriptase (200 U/ul) (Invitrogen).
3. Continue incubation at 42oC for 58-60 min.
4. Add addotopma; 1 ul Superscript RT ( for reaction booster).
5. Incubate at 42oC for additional 1 hr.
6. Add 1.24 ul of 50 mM EDTA (final concentration of 2 mM) (EDTA has been filtered with 0.2 um Disk).
7. Add 1.5 ul of 1 M NaOH (0.2 um filtered).
8. Incubate at 65oC for 30 min.
9. Cool to room temperature.
10. Add 1.5 ul of 1M HCl (0.2 um filtered).

Purification of the Probe using QIAquick PCR purification kit. (modified in our facility)

1. 1. Combine Cy3 and Cy5 reactions.
2. 2. Add 30 ul of sterilized double-distilled water.
3. 3. Add 500 ul Buffer PB.
4. 4. Apply to QIAquick column. Spin at 13000 rpm for 1 min.
5. 5. Re-apply flow-through for optional binding. Spin at 13000 rpm for 1 min.
6. 6. Decant flow-through.
7. 7. Add 750 ul Buffer PE. Spin at 13000 rpm for 1 min. Decant flow-through.
8. 8. Spin at maximum speed for 1 min to dry column.
9. 9. Transfer column into the new 1.5ml tube.
10. 10. Add 30 ul of EB buffer to the center of the membrane. Incubate at room temp for 3 min.
11. 11. Spin at 13000 rpm for 1 min.
12. 12. Repeat elution step with another 30 ul of EB buffer. Pool eluates.
13. 13. Incorporate the 60 ul purified probe into 440 ul of sterilized double-distilled water in an Amicon tube(Millipore).
14. 14. Transfer the mixture into Amicon YM-30 concentrator (Millipore). Cover lid.
15. 15. Place the concentrator into the used Amicon tube.
16. 16. Centrifuge at 10000 rpm for 12-15 min until the probe forms a ring of liquid at the edges of the membrane. Not to dry the membrane completely.
17. 17. Add 23-25 ul of Sigma ArrayHyb buffer onto the concentrator membrane. Pipet up and down a few times to resuspend the probe.
18. 18. Invert the filter and place in a new Amicon tube.
19. 19. Centrifuge at 10000 rpm for 4 min to recover the probe.

Array Hybridization
1. Pre-warm hybridization chamber at 70oC on Slide Moat (Boekel Scientific).
2. To the eluted probe, add 3.5 ul of salmon sperm DNA (5mg/ml stock) and 1.6 ul of tRNA (9.2mg/ml) to block non-specific hybridization.
3. Denature the probe at 95oC for 5 min.
4. Centrifuge at 13000 rpm for 10-20 min to pellet any particulates.
5. Pipet 10-15 ul of water into the wells or gaps located on either end of the hybridization chamber to prevent drying of array during hybridization.
6. Place probe on the center of a cover slip. Cover the probe carefully with the array.
7. Quickly transfer the slide into the hybridization chamber. Tighten the chamber.
8. Incubate the hybridization chamber in 60oC water bath for 3-4 hours.
Aminoallyl RNA labeling
The following Brown's lab protocol is a slight modification* of a protocol developed by Joe DeRisi (UCSF) and Rosetta Inpharmatics (Kirkland, WA). Original document can be obtained at www.microarrays.org.

A. RT Reaction
1. To anneal primer, mix 1-2 ug mRNA with 5 ug of anchored oligo-dT [(dT)₂₀-VN] (Operon, HPLC purified) in a total volume of 18 mL.

oligo dT	5 ug of 2.5 ug/uL	2 uL
mRNA/water	1-2 ug	16 uL

2. Heat to 70C for 10 minutes.  Cool on ice for 5 minutes.

3. Add 11.6 uL of nucleotide mix to each of Cy3 and Cy5 reactions.

Nucleotide Mix for one reaction

5X RT buffer 50X dNTP stock solution DTT Superscript II RT (Gibco) RNasin (Gibco, optional)

0.1M 200U/uL 40U/uL

6.0 uL 0.6 3.0 1.5 0.5

11.6 uL 50X dNTP stock solution using a 4:1 ratio aminoallyl-dUTP to dTTP***:

10 uL each 100 mM dATP, dGTP, dCTP (Pharmacia) 8 uL 100 mM aminoallyl-dUTP** (Sigma, #A0410) 2 uL 100 mM dTTP

**Dissolve 10 mg aminoallyl-dUTP in 170 uL water. Add approx. 6.8 uL 1N NaOH.  Final pH is roughly 7.0 using pH paper.
***Altering the ratio of aminoallyl-dUTP to dTTP will affect the incorporation of Cy dye.
1X dNTP final concentration during labeling

500 uM each dATP, dCTP, dGTP 400 uM aminoallyl-dUTP 100 uM dTTP

4. Incubate reaction for 1 hour at 42C. Add additional 1 uL reverse transcriptase and continue incubation at 42C for an additional 1 hour.

B. Hydrolysis
1. Degrade RNA by addition of 15 uL of 0.1 N NaOH. Incubate at 70C for 10 minutes.
2. Neutralize by addition of 15 uL 0.1 N HCl. To continue with the amino-allyl dye coupling procedure, all Tris must be removed from the reaction to prevent the monofunctional NHS-ester Cy-dyes from coupling to free amine groups in solution.
3. Add 450 uL water to each reaction.

C. Cleanup
Add 500 uL neutralized, diluted reaction mix to a Microcon-30 filter (Amicon). Spin at 12g for 7 minutes. Discard flow through. Repeat process two more times, refilling original filter with 450 uL water. Concentrate to 10 uL. Samples can be stored at -20C indefinitely.

D. Coupling
Add 0.5 uL 1M sodium bicarbonate, pH 9.0 to 50 mM final. Check 1M stock solution periodically for fluctuations in pH. Monofunctional NHS-ester Cy3 (PA23001) and Cy5 dye (PA25001, Amersham) is supplied as a dry pellet. Each tube is sufficient to label 10 reactions under normal conditions. Dissolve dry pellet in 20 uL DMSO. Aliquot 2 uL into 10 single use tubes that are then dried in vacuo and store desiccated at 4C. NHS-ester conjugated Cy dye is rapidly hydrolyzed in water, therefore, do not store in DMSO or water. Decreasing the number of aliquots/dye tube may increase your signal. If you have already made aliquots of dye, simply transfer your cDNA in bicarbonate buffer (10.5 uL) to the aliquot of dye. Alternatively, dissolve Cy dye in 10 ul DMSO and add 1 uL of dye to 10.5 uL of the cDNA reaction. 10% DMSO in the coupling reaction will not affect the chemical reaction. Aliquot unused dye and dry immediately. Incubate 1 hour at RT in the dark. Mix every 15 minutes.

E. Quenching and Cleanup
Before combining Cy3 and Cy5 samples for hybridization, unreactive NHS-ester Cy dye must be quenched to prevent cross coupling. Add 4.5 uL 4M hydroxylamine (Sigma). Let reaction incubate 15 minutes in the dark. To remove unincorporated/quenched Cy dyes, proceed with Qia-Quick PCR purification kit (QIAGEN). Method described below is as specified by manufacturer. Combine Cy3 and Cy5 reactions. Add 70 uL water. Add 500 uL Buffer PB. Apply to Qia-quick column and spin at 13K for 30-60 seconds.(optional: reapply flow-though for optimal binding). Decant flow-through. Add 750 uL Buffer PE and spin 30-60 seconds. Decant flow-through. Spin at high speed to dry column. Transfer spin unit to fresh eppendorf tube. Add 30 uL Buffer EB to center of filter and allow to sit 3 minutes at RT. Spin at 13K rpm for 1 minute. Repeat elution step again with another 30 uL of Buffer EB. Pool eluates. Add 20 uL (20 ug) Cot DNA (Gibco). Add 420 TE and apply to fresh Microcon-30 filter. Spin 12,000g to a volume of 29 uL or less.
For 38 uL array hybridization:
29 uL cDNA probe in TE
1 uL polyA (10 ug; Sigma P9403)
1 uL tRNA (10 ug; Gibco #15401-029)
7 uL 20X SSC
1.2 uL SDS 10%
Heat to 100C for 2 minutes. Let stand 15 minutes RT. Apply 38 uL to 40K array. * Slight modifications to original protocol by Mitch Garber and Anatoly Urisman. RNA amplification (needed when RNA is little), and Labeling should be done after that.

Array Washing
1. Warm the solution (~400mL) of 1X SSC, 0.03% SDS
in microwave until ~60 - 65oC.
2. Pour the warm solution in a slide chamber.
3. Wipe the outside of the hybridization chamber
with paper towel. 4. Unscrew the hybridization chamber. Quickly and carefully transfer the slide with forceps into the slide chamber containing the solution of 1X SSC, 0.03% SDS).5. Agitate the slide chamber gently until the cover slip fall off from the array.
6. Transfer the slide into a Petri dish containing the solution of 1X, 0.03% SDS. Agitate on the orbital shaker for 5 min.
7. Repeat the washing step with 1X SSC, 0.03% SDS for another 5 min.
8. Transfer the slide into a dish containing 0.2X SSC. Agitate on the orbital shaker for 5 min.
9. Transfer the slide into a dish containing 0.05X SSC. Agitate on the orbital shaker for 5 min.
10. Repeat the washing step with fresh 0.05X SSC for another 5 min.
11. Place the slide in the slide rack and spray with 0.05X SSC to prevent uneven drying of the array.
12. Quickly transfer the slide rack onto a 96-well round-bottom lid (up-side-down). Sandwich a kimwipe between the rack and the lid to absorb excess liquid. Place the lid with rack onto the swing bucket rotor of centrifuge for microtiter plates.13. Centrifuge at 650 rpm for 6 min.14. Scan the array immediately or store in a lightproof slide box.

Protein Microarray
Protein profiling
(antibody based)Microarray based
ELISA
Protein-protein
interaction
Peptide microarray
Some examples of microarray projects underway are shown below.

Sarcoplasmic reticulum membrane protein profile. A multiplex approach was used to profile proteins in the sarcoplasmic reticulum of skeletal muscle. Isolated membrane protein fractions were printed and probed with monoclonal antibodies to 7 proteins associated with calcium regulation.

Peptide Array Investigation of Ig-E binding. A peptide array was used to monitor variation in IgE-antibody binding to 15 mer peptides. The sequence corresponding to an epitope was systematically substituted with Alanine.  The set of substituted peptides were printed followed by incubation with sera. The limit of IgE detection was approximately 1 pg.