Biomedical Engineering Reference
In-Depth Information
Important! Avoid loss of solvent!
After cooling, the samples are left for 30 min in an ice bath and are
centrifuged again. The supernatants are used for DNA estimation
by the diphenylamine method (Protocol 1.2.3):
secondsupernatant
.
Add 0.5 - 1.0 ml of Soln. G to the precipitate and heat the mixture
to 90
◦
C for 10 min. After cooling, the samples are centrifuged as
described above and the supernatant is used for protein estimation
by the Lowry protocol (Protocol 1.1.1.1):
third supernatant
.
References
Schmidt G, Thannhauser SJ (1945) J Biol Chem 161:83
1.2.2 Orcin RNA (Ribose) Determination
Solutions/Reagents
A 5mg FeCl
3
or 25 mg FeCl
3
·
6H
2
O in 100 ml conc. HCl
B 100 mg orcin (5-methyl-resorcin) freshly dissolved in 20 ml of
Soln. A
Standard
100
µ
/
g
ml ribose
1.0 ml of the first supernatant (Protocol 1.1.1) is mixed with 2.0 ml
of Soln. B and boiled in a water bath for 30 min. To avoid volume
loss, close the test tubes with glass balls.
After cooling to RT, read the absorbance at 660 nm against
ablank.
1
µ
µ
g RNA.
In protein-free and DNA-free solutions the RNA content is de-
termined by UV measurement at 260 nm.1O.D.
g ribose corresponds to 4.56
µ
≈
0.1
g RNA.
References
Mejbaum W (1939) Z physiol Chem 258:117
1.2.3 Diphenylamine DNA (Deoxyribose) Determination
Solutions/Reagents
A
5% perchloric acid (w/v) in ddH
2
O
B 10% perchloric acid (w/v) in ddH
2
O
C 300 mg diphenylamine are dissolved in 20.0 ml glacial acetic
acid, and then 0.3 ml conc. sulfuric acid are added, followed
by 0.1 ml of 50% aqueous acetaldehyde. This reagent has to by
prepared freshly.
Standard
100
µ
/
g
ml deoxyribose in Soln. A
Mix the Schmidt-Thannhauser powder with 1.0 ml of Soln. A,
and fill up the second supernatant (Protocol 1.1.1) to 1.0 ml with
Soln. B.
Heat the acidic solutions to 70
◦
C for 15 min. After cooling to
RT, mix 1.0 ml of the solutions containing 5 - 10
µ
g deoxyribose
µ
(corresponding to 30 - 60
g DNA) with 2.0 ml of Soln. C, close
with Parafilm and incubate overnight at 30
◦
C.
