Dr. Zamir K. Punja Laboratory

Protocol 1 Ultimate DNA Extraction Protocol Owen (Sep. 2009)

Ultimate DNA extraction protocol

Solutions needed

1. Extraction Buffer For 100 ml
2% CTAB 4 g CTAB
100 mM Tris-HCl pH 8.0 10 ml 1M Tris-HCl pH 8.0
20 mM EDTA 4 ml 0.5M EDTA pH 8.0
5% PvPP (soluble) 5 g PvPP
1.4M NaCl 8.2 g

Buffer is hard to dissolve, you will need to heat it up

Bring to 100 ml with ultra pure water and check the pH.

2. Phenol:Chloroform:Isoamylalcohol (PCI)(50:48:2)

Phenol is mixed with 0.1% w/v hydroxyquinoline
An equal volume of Chloroform:Isoamylalcohol (24:1), store away from light at 4 ° C

3. TE pH 8.0
10 mM Tris-HCl pH 8.0
1 mM EDTA
Must be sterilized

4. 0.5 M DTT
Dissolved in ultrapure water and store at -20 ° C

5. RNAse A solution

Protocol

Freeze dry sample ~200 mg in a 2 ml tube
Homogenize with bead beater 30 s max speed
Add 0.5 of 60 C prewarmed extraction buffer, 5 ul of DTT solution and 1 ul of RNAse A
Heat at 60 C for 30-60 min
Add 500 µl of cold PCI and mix by inverting 6-10 times, centrifuge for 15 min at 9000 rpm
Remove aqueous phase and add to a new 1.5 ml tube, add 500 µl of chloroform:Isoamylalcohol, mix by inversion and spin for 15 min at 9000 rpm
. Remove supernatant and add to a new 1.5 ml tube, add 250 µl isopropanol, mix by inversion incubate at RT for 15 min(no more than 45 min), following incubation centrifuge for 15 min at 14 000 rpm.
. Carefully decant supernatant, wash pellet with 500 µl 70% ethanol, incubate at RT for 15 min (longer is okay, even overnight), following incubation centrifuge at 14 000 rpm for 15 min
. Carefully remove all traces of ethanol and air dry
. When pellet is dry, dissolve in 200 µl of TE, store at -20 ° C

Protocol 2 RNA extraction using Plant RNA isolation reagent (invitrogen) Owen (Sep. 2009)

RNA extraction using Plant RNA isolation reagent (invitrogen)

For difficult to isolate RNA (older/woody tissues) à takes 2 days

Additional reagents needed (need to be RNAse free)

Microcentrifuge tubes

5 M NaCl

10 M LiCl

Chloroform

3M Sodium Acetate

Isopropanol

75% Ethanol

DEPC H₂O

PVPP

Protocol

Small amount of tissue ~0.1-0.2 g FW
Freeze dry, add a pinch of dry PVPP powder and homogenize on bead beater ( 30s max speed)
Add 0.5 ml RNA extraction reagent (careful it is stinky and dangerous), mix on bead beater and let sit at RT for 5 min
Spin at 4 C @12 000 g for 12 min, transfer supernatant to a clean tube
Add 0.1 ml 5M NaCl, and mix by inversion
Add 0.3 ml Chloroform, and mix
Spin at 4 C @12 000 g for 12 min, transfer aqueous phase to a clean tube
Add ¼ vol 10 M LiCl, mix and incubate overnight (or longer) at 4 C
Spin at 4 C @12 000 g for 20 min
Resuspend small pellet in 0.266 ml DEPC H₂O, then add 34 ul NaAC 3M and 0.3 ml Isopropanol, incubate on ice for 15 min
Spin at 4 C @12 000 g for 12 min, wash pellet in 1 ml 75% ethanol
Spin at 4 C @12 000 g for 4 min, carefully remove ethanol, invert tubes and let dry for 10 min, afterwards check them carefully (do not overdry)
Suspend pellet in 30 ul DEPC H₂0
Check concentration on the Nano Drop

Protocol 3 RNA extraction using Yellozol/Trizol from plant material. Owen (Sep. 2009)

RNA extraction using Yellozol/Trizol from plant material.

Preparing Yellozol

Contents For 500 ml

45% Phenol 225 ml Phenol with 0.1% w/v hydroxyquinole

0.8M Guanidine thiocyanate 47.26 g

0.4M Amminioum thiocyanate 15.2 g

10% glycerol 50 ml

0.1 M Sodium Acetate 6.8g

DEPC H2O to volume

pH to 5.0 with Acetic acid/NaOH

Using RNAse free glassware (500 ml graduated cylinder), take Phenol from the organic layer and begin by stirring in the hydroxyquinole. Add the other reagents slowly and carefully, before adjusting pH.

Store at 4 C away from light, good for up to 1 year

RNA extraction

Need the following reagents all reagents need to be RNAse free

Chloroform

DEPC H₂O

Microcentrifuge tubes

75% ethanol

Isopropanol

1.2 M NaCl

Collect sample 100 mg fw/ 1 ml of yellozol is optimal
Freeze dry and homogenize sample on bead beater, glass balls work well for leaf/callus tissue otherwise use steel ball (30s max speed)
Add 1 ml of yellozol, and mix on bead beater 1 min at medium speed (make sure the lids are closed tightly)
Let sit at RT for 2 min
Spin at 4 C @12 000 g for 12 min, transfer supernatant to a clean labeled tube (you can store the tubes here @ -80 C for a year)
Add 0.2 ml of chloroform, mix by inverting and let sit at RT for 3 min
Spin at 4 C @12 000 g for 12 min, transfer aqueous phase to a new clean tube
Add 0.25 ml 1.2 M NaCl and 0.25 ml of isopropanol, mix by inversion and let sit @ RT for 10-15 min (no longer than 15 min)
Spin at 4 C @12 000 g for 12 min, pour off supernatant carefully and wash pellet with 1 ml 75% Ethanol leaving for at least 10 min
Spin at 4 C @5 000 g for 5 min, carefully pull off as much ethanol as possible, invert the tubes on some clean paper towels and let dry for 10 min, after 10 min check the pellets DO NOT OVERDRY
Dissolve in 30-100 ul DEPC H₂O and determine concentration using the Nano Drop, Nano drop can read up to 4 ug/ul if higher you need to dilute samples for readings.