Download II. Conversion Tables and Formulas

Conversion Tables and Formulas
Storage of RNA
Store RNA at –70° to –80°C, as aliquots in ethanol or isopropanol. Most RNA is relatively stable at this temperature. Centrifuge the RNA and resuspend in the appropriate
RNase-free buffer before use.
Drying, dissolving and pipetting RNA
RNA can be dried briefly at 37°C or in a vacuum oven. When working with RNA, place
all samples on ice. For the reasons mentioned above, RNA is very susceptible to degradation when left at room temperature.
Dissolve RNA by adding RNase-free buffer or water, then standing the tube on ice for 15
min. Gently tap the tube or use vortexing with caution.
Temperature sensitivity
Although DNA is relatively stable at elevated temperatures (100°C), most RNA is not
(except for short RNA probes which are stable for 10 min at 100°C).
Therefore avoid high temperatures (>65°C) since these affect the integrity of the RNA.
Instead, to melt out secondary structures, heat RNA to 65°C for 15 min in the presence
of denaturing buffers.
II. Conversion Tables and Formulas
Metric prefixes (International System)
Bigger
Smaller
Symbol
Prefix
Multiplication factor
Symbol
Prefix
Multiplication factor
T
tera
1012
m
milli
10–3
G
giga
10
9
µ
micro
10–6
M
mega
106
n
nano
10–9
k
kilo
103
p
pico
10–12
f
femto
10–15
a
atto
10–18
Moles, molar, molarity
9
Term
Symbol
Meaning
Example
Mole
mol
Absolute amount of
a substance
1 mol  6.022 x 1023 molecules
Molar
or
Molarity
mol/l or M
Concentration of a
substance in a
liquid
1 mol/l  6.022 x 1023 molecules/liter
 a one molar solution  a solution
with a molarity of one
Example of molar equivalence
0.2 pmol in 100 µl means a concentration of 2 nM.
Example how to calculate: 0.2 pmol in 100 µl = 2 pmol in 1 ml = 2 nmol in 1 l.
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Conversion Tables and Formulas
Physical conversions and formulas
Centrifugal force conversion (Dyson, 1991)
RCF = (1.11 x 10–5) (rpm)2 r
RCF = relative centrifugal force (x g; where g = 980 cm x sec–2)
rpm = rounds per minute
r = radius of rotor in mm
Nucleic acid data
Conversions
Spectral constants for nucleotides (Adapted from Sambrook, et al., 1989)
Compound
Molecular weight
(MW)
max (pH 7.0)
Absorbance
at max
(1 M solution)
ATP
507.2
259
15,400
dATP
491.2
259
15,400
CTP
483.2
271
9,000
dCTP
467.2
272
9,100
GTP
523.2
253
13,700
dGTP
507.2
253
13,700
UTP
484.2
260
10,000
dTTP
482.2
267
9,600
Molar concentration of nucleic acid = (observed absorbance at max) ÷ absorbance at
max for 1 M solution.
Spectrophotometric equivalents
1 A260 unit
Nucleic acid
Amount
Molarity
(in nucleotides)
double-stranded DNA
50 µg/ml
0.15 mM
single-stranded DNA
33 µg/ml
0.10 mM
single-stranded RNA
40 µg/ml
0.11 mM
oligonucleotide*
20 – 30 µg/ml
0.06 – 0.09 mM
* For exact determination of the molecular weight, see table “Conversions between weight and molarity of
various DNAs” page 223.
Calculations
9
Determining purity of nucleic acid preparations
For pure DNA:
A260/A280 1.8
For pure RNA:
A260/A280 2.0
An A260/A280 ratio of <1.8 (DNA) or <2.0 (RNA) means the nucleic acid preparation
contains contaminations (e.g., protein), or phenol.
Appendix
221
Conversion Tables and Formulas
Calculating molecular weight of nucleic acids (Adapted from Ausubel et al., 1988)
For molecular weight of
Use this calculation
DNA base pair (sodium salt)
1 base pair = 665 daltons
double-stranded DNA
molecule
(number of base pairs) x (665 daltons/base pair)
single-stranded DNA molecule
(number of bases) x (325 daltons/base)
single-stranded RNA molecule
(number of bases) x (340 daltons/base)
oligonucleotide
For dephosphorylated oligonucleotides:
[(number of A x 312.2) + (number of G x 328.2) +
(number of C x 288.2) + (number of T x 303.2)] – 61
For phosphorylated oligonucleotides:
[(number of A x 312.2) + (number of G x 328.2) +
(number of C x 288.2) + (number of T x 303.2)] + 17
Conversions between picomoles and micrograms of DNA
 For double-stranded DNA (dsDNA):
To convert
Calculate*
pmol to µg
pmol  N 
µg to pmol
µg 
1µg
660pg

= µg
1pmol
106pg
pmol
106pg

= pmol
1µg
660pg
* N = number of base pairs in DNA; 660, average molecular weight of a base pair.
 For single-stranded DNA (ssDNA):
To convert
Calculate*
pmol to µg
pmol  N 
µg to pmol
µg 
1µg
330pg

= µg
pmol
106pg
pmol
106pg
1


= pmol
N
1µg
330pg
* N = number of nucleotides in DNA; 330, average molecular weight of a nucleotide
9
Calculating moles of ends
 Moles of ends for double-stranded DNA molecule:
2 x (grams of DNA) / (MW in daltons)
 Picomoles of ends per microgram of double-stranded DNA:
(2 x 106) / (660 x number of bases)
 Moles of ends generated by restriction endonuclease cleavage, circular DNA
molecule:
2 x (moles of DNA) x (number of sites)
 Moles of ends generated by restriction endonuclease cleavage, linear DNA
molecule:
[2 x (moles of DNA) x (number of sites)] + [2 x (moles of DNA)]
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Conversion Tables and Formulas
Conversions between weight and molarity of various DNAs
Type
Size
pmol/µg
Molecules/µg
oligonucleotide
20 nucleotides
152
µg/pmol
9.1 x 10
13
0.0066
11
0.66
DNA
1000 bp
1.52
9.1 x 10
pUC18/19 DNA
2686 bp
0.57
3.4 x 1011
1.77
pBR322 DNA
4361 bp
0.35
2.1 x 10
11
2.88
M13mp18/19 DNA
7250 bp
0.21
1.3 x 10
11
4.78
48,502 bp
0.03
1.8 x 1010
32.01
 DNA
Protein data
Conversion between DNA and protein
1 kb of DNA = 333 amino acids of coding capacity = a protein of 3.7 x 104 daltons
Conversion between protein molecular weight and absolute weight
100 pmol
MW of protein [kdaltons]
Amount in µg
100
10
50
5
10
1
9
Appendix
223