http://openwetware.org/wiki/Smolke:Colony_PCR_tsb

Overview

Colony PCR from yeast, either to check inserts etc. or for sequencing.

Yeast Colony PCR

1. Pipette 10 ul NaOH (0.02 M) to a pcr tube

2. Take a single colony from your plate and resuspend it in the tube.

3. Boil it in a pcr machine for 10 min at 99 C.

4. centrifuge the tube for few seconds to precipitate cell debries

5. Use 2-3 ul of the supernatant as template in 20 ul pcr reaction.

* For yeast cPCR, I have got better results with Kappa (robust) polymerase in compare to Phusion or Taq specially if fragment size is larger than 500 bp.

*Kappa polymerase reaction mix (*20 μl rxn)

5x KAPA2G Buffer B 4.0 μl
dNTP mix 10 mM 0.40 μl
KAPA2G Robust 0.10 μl
Template genomic DNA (see above) 2-3 ul
Primer forward & reverse
add water to final volume of 20 μl

Travis' Version (Lyticase lysis)

Materials

  • Lyticase (from Sigma)
  • TE
  • PCR buffers, primers, polymerase, etc.

Procedure

The basic idea is breaking the cells with lyticase and heat, then doing PCR.

  1. Dilute stock of lyticase to 50 U/mL in TE.
  2. Aliquot lyticase in 50uL quantities
  3. Pick colonies (I use a pipette tip) and add to lyticase aliquots, pipette up and down or agitate to break up colony
  4. Incubate at 37°C for 30 min
  5. Incubate at 95°C for 10 min
  6. Use as template for PCR - I use 5uL of the cells in a 50uL PCR reaction

Notes

  • I use this protocol to PCR off the chromosome for sequencing... I clean up the rxn with a Zymoclean kit afterwards.
  • The amount of yeast doesn't seem to matter much. I get success with very small colonies or with loads of cells.

Josh's Version (NaOH lysis)

Materials

  • 20 mM NaOH
  • PCR materials

Procedure

  1. Aliquot 20uL NaOH into PCR tubes
  2. Pick colonies (I use pipet tips) into the NaOH
  3. Incubate at 95C for ~45 minutes
  4. Centrifuge at max speed for 10 minutes (helps to use plastic inserts in the microfuge)
  5. Use 1uL of supernatant as template in a (10uL) PCR.

Genomic Prep by Harju Bust'n'Grab

Materials

  • overnight yeast cultures
  • lysis buffer - 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0)
  • phenol:chloroform
  • chloroform
  • ethanol (70% and 100%)
  • sodium acetate
  • TE

Procedure

Adapted by Kate from Harju et al., 2004

  1. Spin down 1.5 ml O/N culture and remove media
  2. Re-suspend in 200 µl lysis buffer and transfer to a thin-walled PCR tube
  3. Freeze at -80 °C, 5 min
  4. Thaw at 95 °C, 1 min on PCR block
  5. Repeat once and transfer to 1.5 mL eppendorf tube
  6. Vortex vigorously
  7. Add 200 µl phenol:chloroform and vortex well
  8. Spin 2 min, remove aqueous phase
  9. Add 200 µl chloroform and vortex well
  10. Spin 2 min, remove aqueous phase
  11. Add 400 µl ice-cold ethanol and 40 µl sodium acetate
  12. Precipitate at -80 °C, 5 min
  13. Spin 5 min at 4 °C
  14. Wash pellet with 70% ethanol
  15. Re-suspend in 50 µl TE - use 1 µl in a PCR reaction

Genomic Prep with Glass Beads

Materials

  • overnight yeast cultures
  • lysis buffer - 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris pH 8.0, 10 mM EDTA
  • phenol:chloroform:isoamyl alcohol
  • ice cold 100% EtOH
  • glass beads

Procedure

Adapted by Mike from protocol from friend in the Herschlag group, and further adapted by Stephanie

  1. Grow 3-10 ml culture of yeast to saturation. (You can even use this protocol on single colonies if you don't need much DNA.)
  2. Collect cells by centrifugation in 1.5 ml eppendorf tube (max speed, 15 s).
  3. Aspirate supernatant and resuspend in 500 µl of ddH2O. Pellet, decant.
  4. Pipette briefly to resuspend pellet in residual liquid.
  5. Add 200 ul detergent lysis buffer, ~100 mg (50uL) glass beads.
  6. Tape to vortex machine in cold room, vortex for 10-20 min. (You can vortex for 2 min at room temp if you don't need much DNA.)
  7. Add 200 ul PCI (phenol:chloroform:isoamyl alcohol; 25:24:1), vortex briefly.
  8. Centrifuge at max for 5 min (4 °C).
  9. Transfer aqueous top layer to fresh eppendorf tube.
  10. Add 50 ul 3M sodium acetate, then 1 ml ice cold 100% EtOH.
  11. Spin for 10-20 min at high speed at 4 °C.
  12. Decant off liquid and vacufuge until pellet dries.
  13. Resuspend in ~100 uL water, 1x TE buffer pH 8.0, or EB buffer.
  14. For PCR use 1 ul of 1:10 or 1:100 dilution of the resuspension as template - this varies depending on the strain, shearing, and how well your genomic isolation went. You can nanodrop the resuspension to estimate the concentration, and use 10-30 ng in a 25 uL PCR reaction.
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