PROTOCOL: CWr
Cell-wall residue preparation
Reagents:
a)
b)
c)
d)
e)
f)
0.1 M acetate buffer, pH 5 (Add acetic acid 0.1M to sodium acetate 0.1M up to pH=5.
Can be store for two weeks at 4ºC)
heat stable amylase (Sigma #A-3403, CAS 9000-85-5)
amyloglucosidase (Roche, #102-857)
95% ethanol (CAS 64175A)
80% ethanol (CAS 64175A)
acetone (CAS 67641A)
Procedure:
=====DAY 1: Starch and Soluble Sugar Removal
(turn waterbaths on to 90 oC and 60 oC)
a) Weigh 100 mg of dried-ground sample into 15ml falcon tube.
b) Add 5 ml COLD acetate buffer and 0.1 ml heat stable amylase. Use small beaker for amylase
and swirl on use.
c) Place in 90 oC waterbath for 1 h and swirl every 20 min.
d) Cool sample to ~50 oC. Make sure samples are cool before proceeding.
e) Add 0.2 ml amyloglucosidase to sample. Swirl bottle of amyloglucosidase and pipette directly
out of suspended solution. Do not pipette off bottom of bottle.
f) Place in 60 oC waterbath for 3 h and swirl every 30 min.
g) Cool sample to ~50 oC.
h) Add 5 ml 95% ethanol and place in refrigerator overnight.
=====DAY 2: Drying
a) Vortex and centrifuge sample at 2800 RPM for 15 min and pour off supernatant.
b) Repeat step a with 5 ml 80% ethanol (2x) and once with 5 ml acetone.
c) Blow off acetone with N2 and breakup pellet by tapping.
d) Just prior to placing in desiccator for minimum of 24 h prior to hydrolysis, break up sample
with glass stir rod.
Cost per 100 samples:
Vol.
5 ml
0.1 ml
0.2 ml
5 ml
10 ml
5 ml
Reagent
Acetate Buffer 0.1M
Amylase
Amyloglucosidase
Ethanol 95%
Ethanol 80%
acetone
N2
Unit Units/sample $/unit $/sample
L
0.005
1.0
0.01
ml
0.1
1.2
0.12
ml
0.2
10.6
2.13
L
0.005
7.2
0.03
L
0.008
7.2
0.06
L
0.005
26.3
0.13
0.20
$2.68
$/100 samples
0.50
12.44
212.64
3.42
5.76
13.13
20.00
$267.88