Gene tagging protocol

PCR reactions were set up using a commercial PCR kit from Fermentas. Reagents from any other supplier should also work. Restriction enzymes were supplied by Fermentas or Roche. Universal checking primers: For checking of bacterial inserts two primers are required: upch-uni located approximately 140 bp upstream of the MCS and dwch-uni located approximately 40 bp downstream of the MCS. For checking of yeast transformants the same primers are used:

  • upch-uni "gaagcagtggtcttcaactcg"
  • dwch-uni "tgtcggggctggcttaacta"
pTAPKan1 is the vector used for cloning tagging constructs. It contains the kanMX6 maker gene, conferring resistance to Geneticin (G418). It was constructed by digestion with NdeI and PacI of pFA6a-TAP-KanMx6 (Kim Nasmyth plasmid collection number 4110) and ligation of two annealed oligonucleotides coding for new restriction sites:
  • TAP_MCSfw aattcatatgatcaccggtaccctaggatccagcgctagcgctgtcgacggtcttaaggcgcccgggttaattaaaatt
  • TAP_MCSrv aattttaattaacccgggcgccttaagaccgtcgacagcgctagcgctggatcctagggtaccggtgatcatatgaatt
The resulting MCS contains the following restriction sites: NdeI - KpnI - BamHI - NheI - SalI - AflII - XmaI - PacI plus other rare ones.
Per ligation you need 10 ng of vector digested with two restriction enzymes and dephosphorylated. It is advisable to prepare the vector very well: Digest it with restriction enzyme, electrophorese it on an agarose gel until the bromphenol blue dye has migrated at least 10 cm, extract it and dephosphorylate it with shrimp alkaline phosphatase.
Bacteria and media:
We used DH5alpha cells made competent using the CaCl2 method and LB medium containing 100 µg/l ampicillin. Other bacterial strains suitable for standard cloning should work just as well. Yeast cells and media: We used S. pombe cells made competent using Li-acetate. Cells was cultured at 32°C in standard YES medium supplemented with 0.15g/l adenine and 0.1g/l uracil, L-histidine, L-lysine and L-leucine. For selection, Geneticin was added at 100 µg/ml. Genomic DNA of S. pombe from which to amplify the homology regions. For DNA purification from PCR, restriction digest or agarose gel we used anion exchange columns from Qiagen. Products of any other supplier should work just as well.

Cloning of the construct

  • Day 1:
    • Run 55 µl PCR reactions with upout+upin and dwout+dwin primers. One reaction contains: 5.5 µl 10x buffer, 4.4 µl dNTPs, 3.3 µl MgCl2, 0.3 µl Taq, 5 µl 5 µM primers and genomic DNA as template. Program: 30x 40 seconds 94 °C, 50 seconds 56 °C, 90 seconds 72 °C.
    • Run 10 µl (+2 µl 6x DNA loading dye) on a 1.5% agarose gel and compare to lanes in the gel-preview.
    • Mix the rest (approx. 40 µl) of both up- and dw- PCRs and purify over a Qiagen gel-extraction kit column. Use 600 µl buffer PB and elute with 30 µl of buffer EB.
    • 1st digest: Add 30 µl containing 2 µl Enz1 restriction enzyme, 5.8 µl 10x buffer and 22.2 µl H2O. Incubate 2 hours at 37 °C.
    • Purify over a Qiagen column. Use 300 µl buffer PB and elute with 30 µl of buffer EB.
    • 1st ligation: Add 30 µl containing 2 µl T4 ligase, 5.8 µl 10x buffer and 22.2 µl H2O. Incubate O/N (16 hours) at 4 °C.
  • Day 2:
    • Purify over a Qiagen column. Use 300 µl buffer PB and elute with 30 µl buffer EB.
    • 2nd digest: Add 30 µl containing 1.5 µl of EnzA restriction enzyme, 1.5 µl of EnzB restriction enzyme, 5.8 µl buffer EB and 21.2 µl H2O. Incubate 2 hours at 37 °C.
    • Add 12 µl 6x DNA loading dye and run 60 µl on a 1% agarose gel. Cut out the target band as indicated on the gel-preview.
    • Gel extract using a Qiagen column. Use 500 µl buffer QG and elute with 30 µl buffer EB.
    • Ligate 5 µl of extracted insert to 10 ng vector using T4 ligase. It is crucial, that the vector is prepared well! Run the appropriately digested vector on a 0.6% TAE agarose gel, let the bromphenol blue dye migrate at least 10 cm, to thoroughly purify the vector. Dephosphorylate the vector with shrimp alkaline phosphatase (can be heat-inactivated). Don't waste your time by rushing this! Do the control without insert!
    • Transform DH5alpha and incubate O/N at 37 °C.
  • Day 3:
    • Prepare four tubes containing 3 ml of LB+Amp medium. Prepare PCR tubes containing 20 µl PCR premix, cool on ice. For 5 reactions (100 µl) use 10 µl 10x buffer, 8 µl dNTPs, 6 µl MgCl2, 0.5 µl Taq, 0.5 µl 100 µM upch-uni primer, 0.5 µl dwch-uni primer and 74.5 µl H2O.
    • Pick four clones with a yellow pipette tip. Dip into the PCR premix and inoculate the LB+Amp culture. Incubate the culture O/N at 37 °C.
    • Run the PCR: 30x 30 seconds 94 °C, 40 seconds 56 °C, 120 seconds 72 °C.
    • Add directly to the PCR tube 5 µl containing 0.5 µl Enz1 restriction enzyme and 4.5 µl H2O. Incubate 2 hours at 37 °C.
    • Add 5 µl 6xDNA loading dye and run 15 µl on a 1.5% agarose gel and compare to gel-preview to identify positive clones.
  • Day 4:
    • Make a miniprep using a Qiagen column from a culture of a positive clone.
    • Digest 4 µl of the miniprep in 14 µl containing 1 µl junction restriction enyzme, 1.4 µl 10x buffer and 7.6 µl H2O for 2 hours at 37 °C.
    • Run 3.5 µl on a gel and transform the rest into yeast.

S. pombe transformation

LiAc solution: 0.1 M LiAc in 1x TE, pH=7.5
PEG solution: 0.1 M LiAc, 40% PEG 3350 in 1x TE, pH=7.5

    Preparation of competent cells:
  • Grow yeast strain in 50 ml YES in 250 ml Erlenmeyer at 32 °C O/N.
  • Next morning, dilute the culture to OD600=0.2 (this is approximately 3 ml of the overnight culture into 100 ml of fresh YES) and incubate 100 ml of the culture in 500 ml Erlenmeyer flask for 4 h at 32 °C until the OD600=0.6.
  • Spin for 3 min 2500 rpm (1000g) in two 50 ml Falcon tubes.
  • Pour off the supernatant and resuspend in 20 ml of LiAc solution and pool the contents of two Falcon tubes.
  • Spin for 3 min 2500 rpm, pour off the supernatant and resuspend in 20 ml of LiAc solution.
  • Spin for 3 min 2500 rpm, pour off the supernatant and resuspend in 2 ml of LiAc solution.
  • Incubate for 30 min at 32 °C.
  • Add to 1,5 ml microfuge tubes: The DNA to be transformed should be in a maximum 15 µl volume. Also include 150 µl of the suspension of competent yeast cells. Vortex briefly.
  • Add 375 µl of PEG solution and 2 µl of single-stranded DNA (we used salmon sperm DNA 10 mg/ml). Mix by inversion.
  • Incubate 45 min at 32 °C.
  • Subject to heat shock by incubating for 5 min at 46 °C in a thermomixer. Let it cool down at room temperature for 10 min.
  • Spin for 30 seconds at 5000 rpm and remove the supernatant using pipette.
  • Resuspend the cells in 100 µl of YES and transfer to a 5 ml snapcap tube containing 1 ml of YES.
  • Incubate O/N at 32 °C with shaking.
  • The next morning, vortex the tubes and plate 200 µl on an appropriate selective plates.
  • Incubate for 3 days at 32 °C.

Checking yeast transformants by PCR

  • For each transformation pick 4 colonies and check the successful TAP-tagging by two colony PCRs. Use primers specific for the target gene ("upch" and "dwch") and two primers specific for the vector ("upch-uni" and "dwch-uni"). Run PCR: 37x 50 seconds 94 °C, 60 seconds 60 °C, 180 seconds 72 °C.
  • Add 15 µl of 2.7x DNA loading dye and run 15 µl on a 1.5% agarose gel. Compare the result to the gel-preview.