Monday, November 10, 2003
Description Genomic DNA Extraction - CTAB Protocol Procedure Place 3 medium-large Arabidopsis rosette leaves in an eppen tube Homogenise leaves in the eppendorf tube Add 500l of 2x CTAB/Mercaptoethanol buffer, keep on ice between samples Incubate samples in 65C water bath for 1 hour Add 500l of Chloroform Mix samples by inverting for 5 minutes at room temperature Centrifuge for 10 minutes at room temperature (15,000 rpm) Collect clear upper layer (400l) in a fresh eppendorf tube Add 250l of isopropanol and invert to mix Incubate at room temperature for at least 10 minutes Centrifuge for 10 minutes at room temperature (15,000 rpm) Discard supernatant (use yellow tips) Add 320l of 1x TE and put samples on ice Ensure pellet is fully re-suspended Add 40l of 1M MgCl2 and invert to mix Incubate on ice for at least 10 minutes Centrifuge for 10 minutes at 4C (15,000 rpm) Collect supernatant in fresh eppendorf tube Add 40l of NaOAc Add 250l of isopropanol Incubate for at least 15 minutes at room temperature Centrifuge for 10 minutes at room temperature (15,000 rpm) Discard supernatant (use blue tips) Add 1 ml of 70% Ethanol and rotate tube to rinse pellet Centrifuge for 5 minutes at room temperature (15,000 rpm) Discard supernatant (use blue tips) Quick spin at room temperature Discard remaining supernatant (use yellow tips) Vacuum dry for 3 minutes Add 50l of 1x TE and leave at room temperature for 10 minutes Store DNA samples at 30C Recipes 2x CTAB buffer (50ml) 1.0g CTAB Powder (=2%) 5.0ml 1M Tris-HCl pH8 (=100mM) 2.0ml 0.5M EDTA (=20mM) 14.0ml 5M NaCl (=1.4M) Make the solution up to 50ml with water CTAB = Cetyltrimethylammonium bromide Also prepare: Add 4ml of 2-Mercaptoethanol to each 1ml of 2xCTAB buffer required Chloroform Isopropanol 1M MgCl2 and 3M NaOAc 70% EtOH 1xTE 3 eppen per sample Set water bath to 65C Supplies Tips (责任编辑:泉水) |