Microsatellite Library Construction

(version 1.0; 01/27/04)

Purpose: create a series of highly polymorphic markers for genotyping of progeny of an experimental genetic cross. These markers will then be ordered into linkage groups according to recombination rates among fish in the cross. The resulting linkage map can be used to identify genetic loci that co-segregate with morphological traits of interest.

Microsatellite Library Construction

Purpose: create a series of highly polymorphic markers for genotyping of progeny of an experimental genetic cross. These markers will then be ordered into linkage groups according to recombination rates among fish in the cross. The resulting linkage map can be used to identify genetic loci that co-segregate with morphological traits of interest.

High Molecular Weight Genomic DNA Isolation [1]

1. Quick freeze an adult fish in liquid nitrogen. Store at ≠80⁰C if material is to be used later

2. Grind into powder with a mortar and pestle, keeping frozen with liquid nitrogen (freeze mortar and pestle first to avoid cracks or fractures)

3. Add powder slowly to extraction buffer in 50 mL conical tube (10 mL buffer per 1 gram starting material; see below for recipe), allow to spread and wet surface of liquid, and then shake tube to submerge

4. Incubate 50⁰C for at least 3 hours (up to overnight)

5. Cool to room temp, add equal volume phenol, and mix gently until emulsion is formed

6. Spin 20 minutes at room temperature at 2000 rpm in a Beckman tabletop centrifuge (~900 x g)

7. Decant and save aqueous (top layer of liquid), repeat steps 5 and 6.

8. Decant and save aqueous, add equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), mix gently until emulsion formed

9. Spin 20 minutes at room temp, at 2000rpm in Beckman tabletop centrifuge

10. Decant and save aqueous, add 0.2 volumes 10M ammonium acetate, 2 volumes ethanol

11. Mix gently to precipitate DNA, and remove DNA using U-shaped pasteur pipette

12. Place DNA in 70% ethanol for 5 minutes

13. Remove 70% ethanol and dry DNA for 5 minutes

14. Resuspend DNA in 5-10 mL of TE (10mM Tris pH 8, 5 mM EDTA) per gram of starting material overnight at room temp

15. Add 100 µg/mL RNaseA (DNAse free) and place at 37⁰C for 3 hours

16. Add equal volume phenol:chloroform:isoamLy alcohol (25:24:1) and mix gently

17. Spin 20 minutes at room temp at 2000 rpm in Beckman tabletop centrifuge

18. Remove aqueous, add 0.2 volumes 10M ammonium acetate, 2 volumes ethanol

19. Mix gently to precipitate DNA, and remove DNA using U-shaped pasteur pipette

20. Place DNA in 70% ethanol for 5 minutes

21. Remove ethanol and dry DNA for 5 minutes

22. Resuspend in 500 mL TE per gram of starting material for 1 hour at 37⁰C

23. Store at 4⁰C

DNA Extraction Buffer

10mM Tris, pH 8.0

100mM EDTA, pH 8.0

0.5% SDS

200 mg/mL proteinase K (Boehringer Mannheim)

Size Selection of Genomic DNA

Blunt end digestion

1. Cut DNA with RsaI (blunt cutter, 4 bp recognition) ≠ do 10 digests for 100 µg total digested

Each digest:

10 µg genomic DNA

1 ml 2 mg/mL RNaseA

1 ml 0.1M spermidine

4 ml 10X buffer 1 (NEB)

4 ml 10U/ml RsaI (NEB)

dH₂0 to 40 ml

2. Digest O/N at 37⁰C, for about 14-18 hours

3. Pool reactions, run 2 µL on 1% TAE gel to verify digestion. To remainder, add 0.1 volume 3M NaOAc (pH 5.2) and 2 volumes ethanol

4. Mix and place on dry ice for 30 minutes

5. Spin 15π in microfuge at 4⁰C on high

6. Decant and wash DNA pellet twice with 70% ethanol (vortex and centrifuge tube on high for 5π after adding ethanol)

7. Decant ethanol and dry pellet until mostly transparent, then resuspend in 50 mL dH₂0

8. Run on 0.8% TAE gel

9. Cut out band in desired size range (800bp-1200bp for sequencing on ABI 377; can use 1000bp-1500bp if using ABI 3730)

10. Gel purify with QIAquick Gel Extraction Kit (Qiagen cat. # 28704)

11. Elute in 30 mL dH₂0 and run 3 ml on 0.8% TAE gel with a mass ladder to check yield

Vector Preparation

Blunt end cloning

1. Digest pBluescript SK(+) with EcoRV (blunt cutter)

10 mg DNA (CsCl prepped)

10 ml 10X buffer 2 (NEB)

1 ml 100X BSA (NEB)

10 mL 20 U/ml EcoRV

dH₂0 to 100 ml

2. Digest 3 hours at 37⁰C (spike at 2.5 hours with 5 ml EcoRV)

3. Run 10 µL of digest with 1 µg uncut on 0.8% TAE gel to check digestion

4. Ethanol precipitate as for genomic DNA digests

5. Resuspend in 85 ml dH₂0

8. Treat with shrimp alkaline phosphatase (SAP)

85 ml DNA

10 ml 10X SAP buffer (USB)

5 ml SAP (USB)

9. Incubate 1.5 hours at 37⁰C

10. Ethanol precipitate as above and resuspend in 40 ml 10 mM Tris pH 7.5 (~250ng/µL)

11. Run 4 µl on 1% TAE gel to check concentration and quality

Ligation and Transformation

1. Ligate insert:vector at 10:1 molar ratio

Controls: no insert, no ligase

Reaction:

proper amount insert

1 µL vector (250 ng)

2 µL 10X ligation buffer (NEB)

2 µL 400U/ml T4 DNA ligase (NEB)

dH₂0 to 20 µL

2. Place at 16⁰C O/N

3. Ethanol precipitate ligations:

1 µL 20 mg/mL glycogen (Boehringer Mannheim)

2 µL 3M NaOAc

40 µL ethanol

Incubate on dry ice for 20 minutes, spin 15π at 4⁰C, wash pellet with 500 µL 70% ethanol

4. Resuspend in 10 µL dH₂0

5. Transform into DH10B electrocompetent cells:

- Chill 1µl DNA, 40µl cells, and 0.2 cm cuvette (Biorad) on ice

- Add cells to DNA on ice, add to cuvette

- Zap with 200 Ohms, 25 µFD, and 2.5 kV

- Add 1 mL SOC to cuvette immediately and transfer to 14 mL round bottom tube

- Grow cells on shaker at 37⁰C for 1 hour

- Spread LB-Ampicillin plates with 25 µl of 40mg/mL Xgal in dimethyformamide

- Plate cells on 10 cm LB-Amp-Xgal plates at low density (~100 colonies per plate)

- Grow O/N at 37⁰C

- Note: grow for no more than 14 hours; otherwise, colonies may be too large

Highly recommended and faster (but much more expensive) alternative: Fermentas Rapid DNA Ligation Kit (cat. # K1422), followed by chemical transformation with Invitrogen One Shot Top10 cells (follow manufacturersπ instructions).

Screening Library by Colony Hybridization

Plating and Lifting Colonies

1. Grow colonies to pinprick size

2. Set up 3 large rectangular Tupperware containers with 2 sheets of Whatman paper at bottom

1- alkali denaturation buffer (1.5M NaCl, 0.5N NaOH)

2- neutralization buffer (1M Tris pH 7.4, 1.5M NaCl)

3- 2X SSC, fill container to ~1 cm (no Whatman)

Cover Whatman in buffer, roll out bubbles with pipette, and save a little excess in container

3. Label Protran (pure nitrocellulose) 82 mm filters with pencil (e.g., filter #1, #2, etc.)

4. Place filters gently on plates, labeled side down

5. Poke through filter and plate with 18-gauge needle dipped in India ink; make several marks in unique pattern around periphery of each plate/filter

6. Place labeled side up on Whatman: 6π in alkali container, 6π in neutralization, then briefly rinse 2X SSC. Gently transfer filter, making sure to avoid bubbles, and wipe excess fluid along side of container as transfer to new container

7. Dry on clean Whatman, 1 hour at RT

8. Dry in oven at 80⁰C (check temp first)

Make stack, alternating Whatman, filter, Whatman, etc.; tape together sides to make compact ≥sandwich≤

After 30 minutes in oven, dry inside glass of oven (remove condensation), dry another 30π, and repeat until no condensation

8. Grow colonies on LB-Amp another 2 hours at 37⁰C, then store at 4⁰C

9. Proceed to hybridization

Hybridization

1. Add prehybridization buffer (i.e., hybridization buffer without probe added) to small round Tupperware and then add filters one by one, allowing each to get wet and sink before adding the next (place up to 20 filters in 100 mL). Incubate at 60⁰C for at least 2 hours

2. Make probes

2 µL DNA (200ng) - di, tri or tetra oligo

8 µL 5X terminal transferase buffer (Promega)

14 µL a³²P-dCTP

1 µL 20 U/ml terminal transferase (Promega)

ddH20 to 40 ml

3. Incubate at 37⁰C for 1 hour

4. Spin Sephadex G50 column (Boehringer Mannheim) for 4π on high (~2500 rpm) in a clinical centrifuge

5. Add probe to column and spin 4π on high into a fresh 2 mL tube

6. Recover probe, check yield on scintillation counter

7. Remove filters from prehyb, place in new Tupperware with 25 mL hybridization solution

8. Add probe to hybridization solution in Tupperware, incubate at 60⁰C O/N

9. Wash filters 0.1X SSC, 0.1% SDS 10π at 60⁰C (more if necessary)

10. Wrap filters in saran wrap and expose to film

Prehybridization and Hybridization Buffer

4X SSPE (for 1 L of 20X stock: 175.3 g NaCl, 27.6 g NaH₂PO₄, 7.4 g EDTA, H₂O to 990 mL; pH to 7.4 with ~6.5 mL 10N NaOH)

1X Denhardtπs Reagent (for 50 mL of 50X stock: 0.5 g Ficoll 400, 0.5 g polyvinilpyrolidone, 0.5 g BSA fr. V, 46.7 mL H₂O)

1% SDS

For 150 mL (enough for one screen of 20 filters):

30 mL 20X SSPE

1.5 mL Denhardtπs

15 mL 10% SDS

103.5 mL ddH₂O

Miniprep and check DNA

1. Pick postive colonies

- Develop film

- Lay film over wrapped filters, trace India ink marks and filter labels (e.g., #1, #2, etc.) on to film; set filters aside

- Lay film on light box, line up plates with film using traced India ink marks; circle positive colonies on plate

2. Grow positive colonies O/N at 37⁰C in 3 mL LB-Amp (50 mg/mL)

3. Prep plasmid using UltraClean Mini Plasmid Prep Kit (MoBio cat. # 12300-100) or other miniprep protocol

4. Elute DNA in 50 ml 10 mM Tris pH 7.5

5. Digest 5 ml DNA to check insert size/DNA quality

5 µL DNA

2 µL 10X buffer 2(NEB)

0.2 µL 100X BSA(NEB)

0.5 µL 20U/µL XbaI(NEB)

0.5 ml 20U/µL XhoI(NEB)

11.8 µL dH₂0

6. Digest at 37⁰C for 2 hours

7. Run digests on 1% TAE gel

Sequencing clones

Sequencing on ABI 377

1. Set up reactions in strip tubes or 96-well plates; for each reaction, use:

x µL DNA (200-500 ng is a good starting point for a 4-5kb plasmid)

1 µL Big Dye v.2 reaction mix

2 µL 1.6 µM primer (T3 or T7)

7.5 µL 2.5x salt buffer (50 mL: 10 mL 1M Tris pH 9, 0.25 mL 1M MgCl₂, 39.75 mL H₂O)

9.5-x µL dH₂0

Note: Keep enzyme and reactions in dark as much as possible

2. Run the following PCR program: 25X (96⁰C-10≤, 50⁰C-5≤, 60⁰C-4π); 4⁰C-hold

3. Store at 4⁰C wrapped in foil until ready to run gel

Sequencing on ABI 3730

1. Set up reactions in strip tubes or 96-well plates; for each reaction, use:

x µL DNA (200-500 ng is a good starting point for a 4-5kb plasmid)

1.5 µL Big Dye v.3.1 reaction mix

2 µL 1.6 µM primer (T3 or T7)

0.75 µL ABI 5x Sequencing Buffer

15.75-x µL dH₂0

Note: Keep enzyme and reactions in dark as much as possible

2. Run the following PCR program: 96∞C-1π; 25X (96⁰C-10≤, 50⁰C-5≤, 60⁰C-4π); 4⁰C-hold

3. Store at 4⁰C wrapped in foil until ready to run gel

Primer design using Primer 3

1. Go to http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi

2. Paste sequence in upper window; be sure ≥pick left primer≤ and ≥pick right primer≤ boxes are selected

3. Type in sequence ID (give sequence a name)

4. Type in a target for the primers to flank; for a 30 bp microsatellite beginning at the 100^th base of your sequence, you would input ≥100, 30≤

5. Change the following default settings:

- product size ranges: 100-300

- primer size: min=18, opt=20, max=22

- primer Tm: min=55, opt=60, max=65

- primer GC%: min=45, max=75

- GC clamp=3 (this parameter is the most flexible; if you do not get results with clamp=3, try progressively smaller clamps)

6. Click ≥Pick Primers≤

7. Save and/or print results; generally, any of the primer outputs should work well.

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[1] Adapted from: Zebrafish Book, Monte Westerfield. (http://zfin.org/zf_info/zfbook/chapt9/9.4.html)