Séraphin Lab
TAP tagging protocol
for yeast
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From the Séraphin lab at the European Molecular Biology Laboratory (EMBL).
Content
Introduction
We use a PCR strategy to generate DNA fragments that are introduced into yeast cells. Integration of this DNA fragment by homologous recombination in the yeast genome fuses the TAP tag downstream of the gene of interest. Transformants are selected using heterologous markers to avoid recombination at the endogenous marker locus (Puig et al., (1998) Yeast, 14, 1139-1146 ,see also Baudin et al. (1993) Nucl. Acids Res., 21, 3329-3330; Langle-Rouault and Jacobs (1995) Nucl. Acids Res., 23, 3079-3081.)
Oligonucleotide design
Two oligonucleotides complementary to the TAP tag-marker cassette at their 3' end and containing the appropriate region of homology with the yeast genome to allow in frame fusion of the TAP tag downstream of the gene of interest are designed (see figure).
PCR amplification of the TAP tag-marker cassette
Prepare on ice the following mix:
- 4 ml of plasmid template (1 ng/ml)
- 12 ml of oligo 1 (10 mM)
- 12 ml of oligo 2 (10 mM)
- 12 ml of dNTPs mix (2.5 mM each)
- 20 ml of Amplitaq buffer
- 139.2 ml of water
- 1 ml of Amplitaq
Divide the mix in 6x30 ml in small PCR tubes (200 ml).
Pre-heat the PCR machine at 94 oC. Insert then the tubes in the machine and run the following program:
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Denature at 94 oC for 5 min.
- 35 cycles of 94 oC for30 sec. denaturation, 48 oC for 30 sec. hybridization and 72 oC extension (1 min.45 sec. with pBS1479, 2 min.10sec. with pBS1539).
- Fill-in at 72 oC for 5 min.
Pool the fractions. Keep 10ml for analysis on a 0.9% agarose gel. Extract the remaining ~170ml with phenol/chloroform/isoamylalcohol.
Add 1/10 volume of sodium acetate 3M pH 4.8. Add 2.5 volume of ethanol 100%. Mix by inverting the tube. Store at -20oC for 1 h.
Spin in cold room at maximum speed for 30 min.
Wash with 900ml of ice cold 70% ethanol (mix by gently inverting the tube).
Spin for 10 min.
Dry in Speedvac for 3 min.
Dissolve in 10ml of TE buffer.
Transformation
Use the10ml solution of PCR fragment obtained above (about 1 mg estimated by gel electrophoresis) for yeast transformation in haploid and/or diploid strains (Ito et al. (1983) J. Bacteriol., 153, 163-168). Select transformants on the appropriate selective plate.
Subclone transformants on selective plate.
Transformant characterization
Grow individual subclones in 2ml of YPD and analyse by western blotting and/or PCR analysis.
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Western blotting
Transfer 1.5 ml of subclones cultures in an Eppendorf tubes.
Spin down the cells.
Add 30ml of glass beads.
Add 100ml of SDS-PAGE loading buffer 1x.
Vortex 3 x 20 sec. storing tubes on ice in between.
Heat 4 min., spin and load 10ml on a mini10% SDS-PAGE gel.
Migrate 1-1.5h at 120 V.
Tranfer the gel on a nitrocellulose menbrane.
Block in Blotto/Tween for at least one hour at room temperature.
Wash 2 x 5 min. with PBS/Tween.
Add 10 ml Blotto/Tween + 5ml of PAP (SIGMA P-2026).
Incubate at RT for 1h.
Rinse 4 x 15 min. with PBS/Tween.
Detect by chemiluminescence.
Remember that the tag contributes ~ 20 kDa.
- PCR analysis
Design two checking oligonucleotides according to figure (oligonucleotide design).
Transfer 1 ml of subclone cultures in Eppendorf tubes. (Do not forget a wild type culture as a negative control).
Spin and remove supernatant.
Resuspend the pellet in 100ml TE/SDS (10 mM TrisCl pH 7.5/1 mM EDTA/3% SDS) (see Polaina and Adam (1991) Nucleic. Acids Res., 19, 5443).
Vortex/shake at room temperature for15 min.
Add 500ml 1xTE.
Extract proteins with 1 volume phenol (Vortex 1 min.).
Recover the upper phase.
Precipitate the DNA with 0.7 volume of isopropanol.
Spin.
Dry the DNA pellet in a Speedvac.
Resuspend the DNA in 20ml of 1xTE.
Use 0.5ml per 20ml PCR reaction.
Prepare on ice:.
- 1.2ml of checking oligo 1 (10mM)
- 1.2ml of checking oligo 2 (10mM)
- 1.2ml of dNTPs mix (2.5 mM each)
- 2ml of Amplitaq buffer (Perkin-Elmer)
- 13.9ml of water
- 0.1ml of Amplitaq (Perkin-Elmer 5 u/ml)
Add 0.5ml of DNA (subclones, wild type control and negative control).
Pre-heat the PCR machine at 94 oC. Insert then the tubes in the machine and run the following program:
- Denature at 94 oC for 5 min.
- 35 cycles of 94 oC for30 sec. denaturation, 48 oC for 30 sec. hybridization and 72 oC extension (use 1 min. per kb).
- Fill-in at 72 oC for 5 min.
Analyze on an agarose gel
Strain storage
Store a correct strain in YPD + 28% sterile glycerol at -80oC.
Reference
Publication reporting results obtained with the TAP strategy should refer to:
A generic protein purification method for protein complex characterization and proteome exploration
Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M. and Séraphin, B.
Nature Biotech., 17, 1030 - 1032 (1999).
[Full text from publisher]
[Abstract from PubMed]
[Comment published in Science (and its Pubmed link)]
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Author: Séraphin@embl-heidelberg.de Last Modified,
August 14, 2000