Genetics and Genomics

scientificprotocols authored over 7 years ago

Author: Marcus Marvin

Nuclei preparation

Preparing the cells

  1. 300–500 ml cultures of density 1.5–4.0 x 10e7 cells/ml
  2. Fix cells with freshly prepared formaldehyde in Buffer A for 2 minutes, stirring, to a final concentration of 1%
  3. Quench fixation by adding glycine to final concentration of 0.125 M
  4. Wash cells twice in Buffer A
  5. Resuspend cells in 5 ml Spheroplasting Buffer and check for lysis

Extract nuclei as follows (taken from Genes to Cells (1996) 1: 475–489)

  1. Place suspension on ice and add 5ml of cold MES Wash Buffer
  2. All subsequent steps were carried out in 4°C.
  3. Collect spheroplasts by centrifugation and wash twice by gentle resuspension and centrifugation in 10ml MES Wash Buffer.
  4. Resuspend in 15 ml MES Lysis Buffer
  5. Lyse spheroplasts with 10 strokes in a homogenizer
  6. Layer the suspension onto a 15 ml sucrose step gradient (5 ml 1.8 M sucrose and 10 ml 1.1 M sucrose, each in the MES Lysis Buffer)
  7. Centrifuge for 10 min at 10000 rpm
  8. Collect intact nuclei, which form a band at the step interface
  9. Determine concentration of nuclei by counting DAPI stained nuclei using fluorescence microscopy.

DNA preparation

  1. For restriction digest, wash nuclei suspension twice with 1x restriction digest buffer
  2. Use 1 x 10e8 nuclei per reaction. The following steps are volumes used per 1 x 10e8 nuclei. Scale up accordingly
  3. Resuspend 1 x 10e8 nuclei in 40 µl 1x restriction buffer
  4. Add SDS to 0.1% final concentration. Incubate for 10 min at 37°C
  5. Add Triton X-100 to 1% final concentration. Mix by pipetting up and down, preventing the formation of bubbles
  6. Add 3 µl of restriction enzyme. Mix and incubate for 90 min
  7. Add SDS to final concentration of 1.6%. Incubate for 20 min at 65°C
  8. Dilute reaction to allow the concentration of DNA to be 2.5 ng/µl
  9. Ligate by adding to final concentrations: 1% Triton X-100, 1x ligation buffer, 1 mg/ml BSA, 10 mM ATP, and 10 µl T4 ligase.
  10. Incubate for 1 h at 16°C.
  11. Add 0.5 µl of 10 mg/ml Proteinase K and incubate overnight at 65°C.
  12. Pool and purify using Qiagen’s PCR purification kit.

Control template

  1. Carry out reaction as above using genomic DNA and digest for with desired restriction enzyme for 4 h
  2. Phenol-chloroform purify the digested genomic DNA
  3. Ligate 300 ng/µl of DNA overnight at 16°C
  4. Phenol-chloroform purify ligated DNA and pool samples


Buffer A

  1. 0.1 M potassium phosphate buffer (pH 6.5)
  2. 5 mM MgCl2

Spheroplasting Buffer

  1. 20 mg/ml yeast lytic enzyme
  2. 25 mM DTT
  3. 1.2 M sorbitol in Buffer A

MES Wash Buffer

  1. 0.1 M MES-NaOH (pH 6.4)
  2. 1.2 M sorbitol
  3. 1 mM EDTA
  4. 0.5 mM MgCl2
  5. protease inhibitors added immediately prior to use

MES Lysis Buffer

  1. 0.1 M MES-NaOH (pH 6.4)
  2. 1 mM EDTA
  3. 0.5 mM MgCl2

protease inhibitors added immediately prior to use


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