1.1 DNA extraction

1.1 DNA extraction kdorfman Fri, 12/16/2011 - 18:34

Wednesday 1/25/2012

Equipment for 1.1

Equipment for 1.1 kdorfman Mon, 12/19/2011 - 16:27

Water bath at 65C
Mortar & pestle
Dry bath at 65C
Centrifuge at 4C
14 mL round bottom POLYPROPYLENE tubes with snap cap. (Becton Dickinson #352059: Fisher 14-959-11B stockroom item)
Paper & scotch tape
Miracloth (EMD_BIO-475855) & funnels
4-8 pots of densely sown Arabidopsis ¹ per pair
Medium weighboat for chopped up leaves
Paper diaper for bench
Liquid N2 + extra ice buckets
Stinky waste bucket in hood

Reagents for 1.1 kdorfman Mon, 12/19/2011 - 17:19

Extracting genomic DNA from Arabidopsis thaliana

DEB

DNA Extraction Buffer

100 mM NaCl
50 mM Tris, pH 8
25 mM EDTA
1% SDS
10 mM beta mercaptoethanol (added at the last minute)

Put exactly 6 mL in each of 13 round bottom centrifuge tubes (see equipment for 1.1). Make sure label on tube says 6 mL.

Add 5 µL BME to each tube just before class

T10E1

Tris 10 mM, EDTA 1 mM, pH 8

50 1 mL aliquots

T10E5

Make 20 0.75 mL aliquots

Ingredient	per pair	per class
DEB	exactly 6 mL in round-bottomed tube	100 mL
	add 5 µL BME per tube at the last minute
T10E1	1 mL	4 mL/pair/semester. Make 100 1mL aliquots
T10E5	1 mL	15 mL
EtOH 95%	1.5 mL	50 mL
EtOH 70%	5 mL	100 mL
ISOprop	exactly 6 mL in round-bottom tube	100 mL
KOAc 5M	4 mL	50 mL
NaOAc 3M pH 5.2	100 µL	5 mL

For	pairs of students
make:	aliquots of exactly 6 mL DEB in round-bottom tubes
make:	1 mL aliquots of T10E1
make:	1 mL aliquots of T10E5
make:	1.5 mL aliquots of EtOH 95%
make:	5 mL aliquots of EtOH 70
make:	exactly 6 mL aliquots of isopropanol in round bottom tubes
make:	4 mL aliquots of KOAc 5M
make:	100 uL aliquots of NaOAc