McClean: Colony PCR (Yeast)

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Revision as of 09:46, 4 October 2011 by Megan N McClean (Talk | contribs)
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Contents

Overview

Our lab's version of yeast colony PCR, adapted from the Botstein Lab's protocol.

Protocol

Reagent Volume
10x HotMaster Taq Buffer with Mg2+ 5μL
10mM dNTP mix 1μL
10μM Primer 1 1μL
10μM Primer 2 1μL
HotMaster Taq DNA polymerase 0.5μL
Sterile Water 40.5μL
Total Volume 50μL

Day 1

  1. Inoculate the strain to transform from a single colony into 5mls of YPD in a test tube. Put on the roller drum at 30°C overnight.

Day 2

  1. Inoculate 50 ml of YPD with 500 μL of the YPD overnight culture in a 250 ml flask. The 500 µl volume is approximate, and depends on the density of the strain you inoculate.
  2. Grow in shaking incubator for about 3-5 hours.
  3. Turn on 42°C water-bath (for heat-shock) if it is not already on.
  4. Harvest the cells by centrifuging in Eppendorf centrifuge model 5810R at 4000rpm (3130 xg) for 5 min. Resuspend pellet in 25 ml of sterile water by vortexing briefly. Pellet again and then resuspend in 1 ml of 100 mM LiAc.
  5. Transfer cell suspension to a 1.5 ml eppendorf tube, centrifuge at 3,000 xg for 2 min in an Eppendorf 5418 centrifuge and discard supernatant by removing it with a pipette.
  6. Add 400 µl 100 mM LiAc and resuspend cells by pipetting up and down. Aliquot 50 μL into 1.5 ml tubes (1 for each transformation). Pellet cells (3,000 xg for 2 min) and remove supernatant by aspiration.
  7. Add 300 μL T mix to each eppendorf tube of cells. Per one transformation reaction add IN ORDER:
    • 240 μL 50% PEG 3350
    • 35 μL 1.0 M lithium acetate
    • 25 μL 2 mg/ml sssDNA
    • 50 μL sterile H20 and 20 μL of DNA (Note: You are aiming for a final concentration between 0.1-10 μg for plasmid DNA. Adjust your DNA and water amounts to add 70 μL of volume total)
  8. Vortex to resuspend cells.
  9. Incubate for 30 minutes at 30°C.
  10. Incubate tubes in a water bath at 42°C for 20-25 (up to 40) min. The time may need to optimized for your strain and transformation conditions.
  11. Microfuge at 3,000 xg for 15s, and remove transformation mix with a micropipette. (NOTE: If you are transforming cells with a drug resistance marker such as KanMX, NatMX, HygMX or selecting for 5-FOA resistance, DO NOT plate your cells now, you need to do a recovery step. See below.)
  12. Add 200 µL of sterile water to each tube and resuspend cells by pipetting it up and down as gently as possible if high efficiency is important.
  13. Plate your cells using glass beads to spread the cells. Add 3-4 glass beads to each plate that you will be using, add about 200μL of cells + water, and spread by shaking the plate horizontally. To ensure single colonies:
    • Plate 150 µl of sterile water and add 20 µl cell suspension in one selection plate #1.
    • Plate the remaining 180 µl in selection plate #2
  14. Incubate at 30 °C. Colonies should appear after 2-4 days.

Recovery step for drug resistance markers and 5-FOA

  • If you are plating your cells onto plates with G418, clonNat, hygromycin, or 5-FOA (basically if you are trying to select for anything BUT ability to grow without a particular amino acid) you need to give your cells some time to recover and express the resistance marker after you've transformed them. This is done after you have removed the transformation mix but before you plate the cells. You have two options for recovery:
  1. Gently resuspend cells in 1ml of YPD. Put this tube at 30°C for 1-4 hours (with a tube clamp to keep the eppendorf from popping open). After recovery, spin down the cells, resuspend in sterile water, and plate as above.
  2. Gently resuspend cells in 1ml of YPD. Plate 200μL cuture onto a YPD plate. In the morning, replica plate this lawn of cells onto a selection plate. You should see colonies in 2-4 days. We usually save the rest of the cells resuspended in YPD overnight at 30°C (with a tube clamp to keep the eppendorf from popping open) and plate about 200μL of this onto a selection plate in the morning just as a back-up.
  • We have found that plating onto YPD and replica plating the next day gives the best results. For 5-FOA transformations it seems crucial to do it this way, as allowing the cells to grow in liquid YPD for any amount of time allows cells with mutations in URA3 to arise and these are able to grow on 5-FOA.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

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References

Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96.

Contact

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