We use the same composition of extraction buffer without dilution.

Thank you, Mahima

I will try to do without dilution and update the result.

Hyunsook Moon,

We use 2 different protocols

one fast (1)and good for large amount of material of good quality,

and another (2) most reliable for small amount of samples or complicated tissues. This second protocol is so good that this DNA is suitable for qPCR

(1). extraction buffer:

Tris-HCl pH8 200mM

NaCl 250mM

EDTA (pH8) 25mM

SDS 0,5 % P/V.

Extraction protocol:

a- Mortar in an epp with pestle a 10-30mg of a young leaf

b- Add 600ul de extraction buffer

c- Centrifuge 5min 12000xg

d- Transfer the supernatant to a new tube and add 0.8 vol of Isopropanol (500ul approx.)

e- Centrifuge 5min 12000xg at 4ºC.

f- Discard the SN and dry at RT for 10-20min until no Alcohol odor.

g- Add 50ul of DNAse free water.

h- Incubate 5min at 65ºC

Use 1/2ul for PCR

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(2) Extraction buffer:

Tris-HCl pH8 100mM

NaCl 1.4M

EDTA (pH8) 20mM

CTAB (detergent) 2% P/V.

B-mercarptoethanol 0.2% V/V

Extraction protocol

a- Mortar in an epp with pestle a 2-100mg of tissue with liquid nitrogen

b- Add 600ul de extraction buffer, shake it vigorously

c- Incubate 30min at 65ºC, shake it vigorously each 10min

d- Add 1vol of chloroform/ isoamyl alcohol (24:1 V:V) shake it vigorously

e- Centrifuge 10min 5000xg

f- Transfer the supernatant to a new tube and add 0.6 vol of cold Isopropanol (400ul approx.) mix by inversion

i- Centrifuge 15min 12000xg at 4ºC

g- Discard the SN

j- Add 600ul of cold EtOH 70% and Centrifuge 5min 12000xg at 4ºC

h- Discard the SN, carefully

i- Dry at RT for 10-20min until no Alcohol odor.

j- Add 50ul of DNAse free water.

k- Incubate 5min at 65ºC

Use 1/2ul for PCR

Best regards!