Author: Marcus Marvin ### Nuclei preparation **Preparing the cells** 1. 300–500 ml cultures of density 1.5–4.0 x 10e7 cells/ml - Fix cells with freshly prepared formaldehyde in Buffer A for 2 minutes, stirring, to a final concentration of 1% - Quench fixation by adding glycine to final concentration of 0.125 M - Wash cells twice in Buffer A - 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 - All subsequent steps were carried out in 4°C. - Collect spheroplasts by centrifugation and wash twice by gentle resuspension and centrifugation in 10ml MES Wash Buffer. - Resuspend in 15 ml MES Lysis Buffer - Lyse spheroplasts with 10 strokes in a homogenizer - 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) - Centrifuge for 10 min at 10000 rpm - Collect intact nuclei, which form a band at the step interface - 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 - Use 1 x 10e8 nuclei per reaction. The following steps are volumes used per 1 x 10e8 nuclei. Scale up accordingly - Resuspend 1 x 10e8 nuclei in 40 µl 1x restriction buffer - Add SDS to 0.1% final concentration. Incubate for 10 min at 37°C - Add Triton X-100 to 1% final concentration. Mix by pipetting up and down, preventing the formation of bubbles - Add 3 µl of restriction enzyme. Mix and incubate for 90 min - Add SDS to final concentration of 1.6%. Incubate for 20 min at 65°C - Dilute reaction to allow the concentration of DNA to be 2.5 ng/µl - 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. - Incubate for 1 h at 16°C. - Add 0.5 µl of 10 mg/ml Proteinase K and incubate overnight at 65°C. - 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 - Phenol-chloroform purify the digested genomic DNA - Ligate 300 ng/µl of DNA overnight at 16°C - Phenol-chloroform purify ligated DNA and pool samples ### Buffers **Buffer A** 1. 0.1 M potassium phosphate buffer (pH 6.5) - 5 mM MgCl2 **Spheroplasting Buffer** 1. 20 mg/ml yeast lytic enzyme - 25 mM DTT - 1.2 M sorbitol in Buffer A **MES Wash Buffer** 1. 0.1 M MES-NaOH (pH 6.4) - 1.2 M sorbitol - 1 mM EDTA - 0.5 mM MgCl2 - protease inhibitors added immediately prior to use **MES Lysis Buffer** 1. 0.1 M MES-NaOH (pH 6.4) - 1 mM EDTA - 0.5 mM MgCl2 protease inhibitors added immediately prior to use