1.2 DNA Quantification

"Dilution Buffer"

10 mM Tris, pH 7.5

Dilute from 1M stock

1 mL stock + 99 mL sterile H2O

OR use T10E1

They need ~600 µL for dilutions and 100 for a spectrophotometric blank. They can take another aliquot from the rack.

"Running Buffer"

dilute from 50X stock

2 gels per pair x 300 mL per gel x 10 pairs = 6 L

Make 8 L

Add EtBr 1 µL/100 mL

50 mL/gel x 2 gels/pair x (10 pairs + 4 extra) = 1400 mL

make 2 batches, one without RNAse and one with

DO THE RNASE-FREE FIRST so the RNAse does not contaminate the RNAse-free agarose! Label the tubes!

1400 mL 1X TAE (=28 mL 50X + water to 1400 mL)

700 mL in each of two flasks marked Etbr

6.3 g agarose in each flask (Multi-purpose agarose from Krackeler)

microwave 1 loosely capped bottle at a time at 50% power ~ 8 min

when cool enough to handle, add EtBr (7µL of 10mg/mL stock) to each flask
1 µL EtBr/100 mL
(7µL x 10mg/mL)/700 mL = 0.0001mg/mL = 0.1µg/mL

To one bottle only:

Add 3.5µL of 20mg/mL RNAseA stock to final conc 0.0001mg/mL

0.005 µL RNAseA / mL TAE

(3.5µL x 20mg/mL)/700mL = 0.0001mg/mL

NOTE: some RNaseA stocks are 10mg/mL

Aliquot into 50 mL sterile conical centrifuge tubes

gels:
%:
TAE:	mL
agarose:	g
EtBr:	µL

*Add weighed agarose to measured TAE in a flask (about half the maximum volume of the flask)

*Boil in the microwave CAREFULLY (power level 0.5) until completely dissolved (check by swirling) DO NOT LET IT BOIL OVER

*Allow to cool slightly

*Add EtBr (in the fume hood)

*Aliquot into 50 mL conical tubes

*Put in rack in 65C waterbath

To make more RNaseA stock from 25 mg lyophilized powder:

For 10mg/mL:

Dissolve in 2.5mL of 10mM Tris-HCl 15mM NaCl, pH 7.5

Heat to 100 º C 15 min, then cool slowly.

Dispense into aliquots and store at -20 µC

For 20 mg/mL: 2.5 mg RNaseA in 1.25mL