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CTAB DNA Extraction Method

The document outlines a protocol for CTAB DNA extraction, detailing the required extraction buffer components and their concentrations. It provides step-by-step instructions for harvesting plant tissue, incubating, centrifuging, and purifying DNA. The final product is to be dissolved in TE buffer or nuclease-free water and stored at -20°C.

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bidhanmalakar2015
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124 views1 page

CTAB DNA Extraction Method

The document outlines a protocol for CTAB DNA extraction, detailing the required extraction buffer components and their concentrations. It provides step-by-step instructions for harvesting plant tissue, incubating, centrifuging, and purifying DNA. The final product is to be dissolved in TE buffer or nuclease-free water and stored at -20°C.

Uploaded by

bidhanmalakar2015
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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CTAB DNA Extraction

Extraction Buffer:
Required vol. Working conc.
H2O 5.8 ml
5M NaCl 2.8 ml 1.4M
500mM EDTA 0.4 ml 20mM
1M Tris (pH=8) 1.0 ml 100mM
CTAB powder 200mg 2%
Β-ME 20ul 0.2%
(NOTE: warm the solution before adding CTAB to dissolve it, vortex thoroughly. Add β-ME just
before use)

Protocol:
1. Harvest a young rosette leaf into liq. N2.
2. Grind the tissue in a 1.5ml tube with pestle and add o.5ml extraction buffer. Keep all
samples on ice until all samples are ready for next step.
3. Incubate samples at 60°C for 30 mins. (use dry bath or wet bath).
4. C/N at 13K rpm for 10 mins.
5. Transfer sup. to another tube.
6. Add 2ul RNaseA (10mg/ml) (DNase and Protease free) and incubate at 37° C for 30
mins.
7. Add 0.5ml of phenol: CHCL3: iso-amyl alcohol (25:24:1) and mix by inverting the
tube gently to avoid shearing of DNA.
8. C/N at 13K rpm for 10mins.
9. Transfer the upper aqueous to another tube.
10. Add 1ul of glycogen, 55ul 3M Na acetate pH – 5.6 and 0.55ml isopropanol.
11. Invert the tube gently to mix and leave at -20°C for 60 mins.
12. C/N at 13K rpm for 20 mins and then remove the sup.
13. Wash the pellet with 0.5ml 70% ethanol and C/N at 13K rpm for 5 mins.
14. Remove the wash and air dry the pellet, don’t allow it to dry too much otherwise
DNA will not dissolve easily.
15. Dissolve the pellet in 100ul of TE buffer or nuclease free water, store at -20°C

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