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



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