Core Bioreagents

Technical Library

Buffer pKa/pH Tables and Formulas:
Useful pH Ranges of Selected Biological Buffers (25 °C, 0.1M)
Trizma Buffer Table - pH vs. Temperature
Phosphate Buffer Table - 0.2M solution
Commonly Used Electrophoresis Buffers
Helpful Formulas

DNA / Protein Electrophoresis and Troubleshooting Tables:
Effective Range of Separation of DNA in Polyacrylamide Gels
Effective Range of Separation of DNA in Agarose Gels
DNA Size Migration of Sample Loading Dyes
Troubleshooting Guide for SDS-PAGE Protein Electrophoresis

Genetic Markers Tables:
Significant Genetic Markers in Commonly Used E. coli Strains
Other Genetic Markers that Affect Growth Requirements in Commonly Used E. coli Strains

RNase Contamination Prevention:
Quick Reference Guide for Preventing RNase Contamination

Nucleotide / Amino Acids Properties:
Nucleotide Physical Properties
Amino Acids Table

Life Science Conversions:
Mass to Moles Conversions
Molecular Masses of Nucleic Acids
Protein Conversion Data Concentration of Protein to Absorbance of Protein
Mass of Protein to Mole of Protein



Buffer pKa/pH Tables and Formulas:

Useful pH Ranges of Selected Biological Buffers (25 °C, 0.1M)

Buffers Useful pH Range pKa
(at 20)
pKa
(at 25)
pKa
(at 37)
Reagent Grade BPC
Grade
Ultra
Grade
MES 5.5-6.7 6.16 6.10 5.97 M8250 M2933 M5287
Bis-Tris 5.8-7.2 n/a 6.50 6.36 B9754 B4429 B7535
ADA 6.0-7.2 6.65 6.59 6.46 A9883 n/a A8074
aces 6.1-7.5 6.88 6.78 6.54 A9758 A3594 A7949
PIPES 6.1-7.5 6.80 6.76 6.66 P6757 P1851 P8203
MOPSO 6.2-7.6 n/a 6.90 6.75 M8389 n/a n/a
Bis-Tris Propane 6.3-9.5 n/a 6.8, 9.0 n/a B6755 B4679 B9410
BES 6.4-7.8 7.17 7.09 6.90 B9879 B4554 B6420
MOPS 6.5-7.9 7.28 7.20 7.02 M1254 M3183 M5162
TES 6.8-8.2 7.50 7.40 7.16 T1375 T5691 T6541
HEPES 6.8-8.2 7.55 7.48 7.31 H3375 H4034 H7273
DIPSO 7.0-8.2 n/a 7.60 7.35 D9648 n/a D0306
MOBS 6.9-8.3 n/a 7.60 n/a M3295 n/a n/a
TAPSO 7.0-8.2 n/a 7.60 7.39 T9269 T5566 T0432
Trizma 7.0-9.0 8.20 8.06 7.72 T1503 T6066 T6791
HEPPSO 7.1-8.5 n/a 7.80 6.66 H3137 n/a n/a
POPSO 7.2-8.5 n/a 7.80 7.63 P3405 n/a P7088
TEA 7.3-8.3 n/a 7.80 n/a T1377 n/a n/a
EPPS 7.3-8.7 n/a 8.00 n/a E9502 E0276 E1894
Tricine 7.4-8.8 8.16 8.05 7.80 T0377 T5816 T9784
Gly-Gly 7.5-8.9 n/a 8.20 n/a G1002 G3915 G7278
Bicine 7.6-9.0 8.35 8.26 8.04 B3876 n/a B8660
HEPBS 7.6-9.0 n/a 8.30 n/a H6903 n/a n/a
TAPS 7.7-9.1 8.49 8.40 8.18 T5130 T5316 T9659
AMPD 7.8-9.7 n/a 8.80 n/a A9754 n/a A9074
TABS 8.2-9.6 n/a 8.90 n/a T1302 n/a n/a
AMPSO 8.3-9.7 n/a 9.00 9.10 A6659 n/a A7585
CHES 8.6-10.0 9.55 9.49 9.36 C2885 n/a C8210
CAPSO 8.9-10.3 n/a 9.60 9.43 C2278 n/a C8085
AMP 9.0-10.5 n/a 9.70 n/a A9879 n/a A9199
CAPS 9.7-11.1 10.56 10.40 10.02 C2632 n/a C6070
CABS 10.0-11.4 n/a 10.70 n/a C5580 n/a n/a
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Trizma Buffer Table - pH vs. Temperature

pH at Temperature   g/L for 0.05 M Solution
5°C 25°C 37°C   Trizma HCL Trizma Base
7.76 7.20 6.91   7.02 0.67
7.89 7.30 7.02   6.85 0.80
7.97 7.40 7.12   6.61 0.97
8.07 7.50 7.22   6.35 1.18
8.26 7.70 7.40   5.72 1.66
8.37 7.80 7.52   5.32 1.97
8.48 7.90 7.62   4.88 2.30
8.58 8.00 7.71   4.44 2.65
8.68 8.10 7.80   4.02 2.97
8.78 8.20 7.91   3.54 3.34
8.88 8.30 8.01   3.07 3.70
8.98 8.40 8.10   2.64 4.03
9.09 8.50 8.22   2.21 4.36
9.18 8.60 8.31   1.83 4.65
9.28 8.70 8.42   1.50 4.90
9.36 8.80 8.51   1.23 5.13
9.47 8.90 8.62   0.96 5.32
9.56 9.00 8.70   0.76 5.47
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Phosphate Buffer Table - 0.2M solution

Sodium Phosphate Monobasic Anhydrous g/L Sodium Phosphate Dibasic Heptahydrate g/L 23°C pH   Sodium Phosphate Monobasic Anhydrous g/L Sodium Phosphate
Dibasic Heptahydrate g/L
23°C pH
22.4 3.49 5.7   10.80 29.51 6.9
22.08 4.29 5.8   9.36 32.73 7.0
21.60 5.37 5.9   7.92 35.95 7.1
21.05 6.60 6.0   6.72 38.63 7.2
20.40 8.05 6.1   5.52 41.31 7.3
19.56 9.93 6.2   4.56 43.46 7.4
18.60 12.07 6.3   3.84 45.07 7.5
17.64 14.22 6.4   3.12 46.68 7.6
16.44 16.90 6.5   2.52 48.55 7.7
15.00 20.12 6.6   2.04 49.09 7.8
13.56 23.34 6.7   1.68 49.89 7.9
12.24 26.29 6.8   1.27 50.81 8.0
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Commonly Used Electrophoresis Buffers
Tris-Acetate (TAE) 1X 0.4 M Tris-Acetate/0.001 M EDTA, pH 8.3
Tris-Phosphate (TPE) 1X 0.08 M Tris-Phosphate/0.002 M EDTA, pH 8.0
Tris-Borate (TBE) 1X 0.089 M Tris-Borate/0.002 M EDTA, pH 8.3

Helpful Formulas

Percentage by weight (w/v)
(% buffer desired / 100) x final buffer volume (ml) = g of starting material needed.
Molar Solutions
desired molarity x formula weight x solution final volume (L) = grams needed
Henderson-Hasselbach Equation
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DNA / Protein Electrophoresis and Troubleshooting Tables
 

Effective Range of Separation of DNA in Polyacrylamide Gels
% Acrylamide (w/v)¹ Efficient Range of Separation (bp)
3.5 1000-2000
5.0 80-500
8.0 60-400
12.0 40-200
15.0 25-150
20.0 6-100
1) N,N'-methylenebisacrylamide is included at 1/30th the concentration of acrylamide.

Effective Range of Separation of DNA in Agarose Gels
% Agarose (w/v) Efficient Range of Separation of Linear DNA Molecules (kb)
0.3 5-60
0.6 1-20
0.7 0.8-10
0.9 0.5-7
1.2 0.4-6
1.5 0.2-3
2.0 0.1-2

DNA Size Migration of Sample Loading Dyes
Agarose Concentration (%w/v) Xylene Cyanole Bromophenol Blue
0.1-1.5 4-5 kb 400-500 bp
2.0-3.0 (sieving agarose) 750 bp 100 bp
4.0-5.0 (sieving agarose) 125 bp 25 bp

Troubleshooting Guide for SDS-PAGE Protein Electrophoresis
Problem Possible causes Solution
Faint or missing protein bands Load quantity is below the detection level of the stain Check the A280 and increase sample concentration
    Use a more sensitive stain (e.g. silver stain)
  Proteins were not fixed in the gel Use a stain which also fixes the proteins
    Use gel fixing solution
  Small peptides (<4 kDa) did not fix in the gel Fix the gel with 5% glutaraldehyde. Rinse the gel well with water before staining
  Proteins are degraded Check the A280 and avoid protease contamination
Avoid freeze-thaw of samples
  Protein ran off the gel Use a higher concentration PAGE gel. See precast gels for recommended gel concentration or use a 4-20% gel if the size is unknown
Film on gel after staining Precipitated Coomassie Blue R Rinse the gel for 15 seconds in methanol and immediately return to water or destain
Poor band resolution Concentration of protein is high Load 10 μg per protein or 100 μg per protein extract
  Age of the gel, due to base catalyzed
hydrolysis of the amide
Order fresh precast gels or cast a fresh gel
  Improper gel concentration See precast gels for recommended gel concentration or use a 4-20% gel if the size is unknown
Band smearing High salt concentrations Dialyze sample, precipitate the protein with TCA or use desalting columns
  Concentration of protein is high Load 10 μg per protein or 100 μg per protein extract
  Protein aggregation Add 4-8 M urea to the sample
Add fresh DTT (30 mM) or 2-mercaptoethanol (5%)
  Voltage is high Electrophorese at 10-15 V/cm
Protein precipitation in the well Hydrophobic proteins Add 4-8 M urea to the sample
White precipitate in sample SDS precipitation Could be due to the presence of guanidine or potassium salts in the sample

Genetic Markers Tables
 

Significant Genetic Markers in Commonly Used E. coli Strains
Marker Description Significance
dam Endogenous adenine methylation at GATC sequences abolished High recombination frequency, constitutively express DNA repair functions. Makes DNA susceptible to cleavage by certain restriction enzymes
deoR Regulatory gene that allows for constitutive synthesis of genes involved in deoxyribose synthesis Allows the uptake of large plasmids
dnaj One of several chaperonins inactive Stabilizes certain mutant proteins expressed in E.coli
endA1 Endonuclease mutation Absence of endonuclease improves the quality and yield of plasmid DNA
F' F' episome, male E. coli host Necessary for M13 infection
gyrA Mutation in DNA gyrase Confers resistance to nalidixic acid
laclq Overproduces the lac repressor Overproduces the lac repressor protein, which regulates transcription from the lac promoter
lacZΔM15 Partial deletion of lacZ that allows α-complementation Required for use with pUC or M13 vectors. Results in blue and white colonies or plaques for clone selection when plated on X-gal
ΔIon Deletion of the Ion protease Reduces degradation of β-galactosidase fusion proteins to enhance antibody screening of φ libraries
mcrB Mutation eliminating restriction of DNA methylated at the sequence 5'-GmC-3' Absence of the mcrB gene product allows more efficient cloning of DNA containing 5-methylcytosine or 5-hydroxymethylcytosine
mrr Mutation eliminating restriction of DNA methylated at the sequence 5'-CmAG-3' Absence of the mrr gene product allows more efficient cloning of DNA containing methyladenine residues
recA Homologous recombination abolished Useful when working with sequences containing direct repeats >50 bp
recB, recC Exonuclease and recombination activity of Exonuclease V abolished Stability of inverted repeat sequences enhanced
recD Exonuclease activity of ExoV abolished Inverted repeat sequences in λ can be propagated
supE/supF Amber suppressors Needed to grow amber mutants
Tn5 Transposon Encodes resistance to the antibiotic kanamycin
Tn10 Transposon Encodes resistance to the antibiotic tetracycline

Other Genetic Markers that Affect Growth Requirements in Commonly used E. coli Strains
Marker Effect on Growth
IeuB Requires leucine
metB Requires methionine
proA/B Requires proline
thi-1 Requires thiamine
galK/U Cannot metabolize galactose
mtlA Cannot metabolize mannitol
xyl5 Cannot metabolize xylose
nupG Alters nucleoside uptake

RNase Contamination Prevention
 

Quick Reference Guide for Preventing RNase Contamination
Precautions Methods Reason Hints
General lab space See below To create an RNase-free area in which to work with RNA All equipment in this area should be RNase-free.
Everything should be handled with gloves and wiped down with 70% ethanol and DEPC treated water before and after use.
Equipment Wiped down with 70% ethanol and DEPC treated water To prevent contamination of samples from local and airborne RNases All equipment in this area should be RNase free. Everything should be handled with gloves and wiped down with 70% ethanol and DEPC treated water before and after use.
Gloves   RNase present in oil on hands Change gloves often as doorknobs, micropipettors,
and refrigerator door handles may be contaminated.
Glass Bake in a dry heat oven
3-4 hours at 180-200 °C
Inactivates RNase Effective method of purging glassware of RNase activity. Some labs set aside equipment that will be used exclusively for RNA work: gel boxes, pipettors, and glassware.
Metalware Bake in a dry heat oven 3-4 hours at 180-200 °C   Effective method of purging heat resistant
equipment of RNase activity.
Plasticware Autoclave in small batches for RNA use only Continuous handling of plasticware can lead to RNase contamination Use individually wrapped sterile products. Always
handle RNase free tubes (conical and microcentrifuge) with gloves.
Polycarbonate and
polystyrene equipment
3% hydrogen peroxide   Soak in a 3% hydrogen peroxide solution for 10 minutes and then rinse thoroughly with RNase free water (see below).
Solutions DEPC Chemical RNase inhibitor (not compatible with polycarbonate or polystyrene) Use at 0.1% in water. Incubate for several hours and autoclave after treatment in order to destroy remaining DEPC. (Note: Do not add DEPC to any buffers containing Trizma or mercaptans. DEPC is reactive with these products. Instead use DEPC treated water to make up Trizma containing buffers.)
1. Farrell, R.E. (1993) RNA Methodologies: A Laboratory Guide for Isolation and Characterization. Academic Press, San Diego, CA 33-39
2. Blumberg, D.D., Creating a ribonuclease-free environment. Methods in Enzymol., 152, 20-24 (1987).
3. Sambrook, J., et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY.

Nucleotide / Amino Acids Properites


Nucleotide Physical Properties
  Nucleotide Molecular Weight (Free Acid) λmax (pH 7) εm at λmax (pH 7)
ATP 507.2 259 15,400
CTP 483.2 271 9,000
GTP 523.2 253 13,700
UTP 484.2 262 10,000
dATP 491.2 260 15,100
dCTP 467.2 270 9,100
dGTP 507.2 253 13,800*
TTP 482.2 267 9,600
*εm value for deoxyguanosine monophosphate (dGMP)
Reference: Specifications and Criteria for Biochemical Compounds, 3 Ed. Sigma Chemical Company (St. Louis, MO: 1984)
Amino Acids Table
Amino Acid Three-Letter Code Single-Letter Code Formula MW
Alanine Ala A C3H7NO2 89.1
Arginine Arg R C6H14N4O2 174.2
Asparagine Asn N C4H8N2O3 132.1
Aspartic Acid Asp D C4H7NO4 133.1
Cysteine Cys C C3H7NO2S 121.2
Glutamic Acid Glu E C5H9NO4 147.1
Glutamine Gln Q C5H10N2O3 146.1
Glycine Gly G C2H5NO2 75.1
Histidine His H C6H9N3O2 155.2
Isoleucine Ile I C6H13NO2 131.2
Leucine Leu L C6H13NO2 131.2
Lysine Lys K C6H14N2O2 146.2
Methionine Met M C5H11NO2S 149.2
Phenylalanine Phe F C9H11NO2 165.2
Proline Pro P C5H9NO2 115.2
Serine Ser S C3H7NO3 105.1
Thereonine Thr T C4H9NO3 119.1
Tryptophan Trp W C11H12N2O2 204.2
Tyrosine Tyr Y C9H11NO3 181.2
Valine Val V C5H11NO2 117.1

Life Science Conversions 
 

Mass to Moles Conversions
1 kb DNA Fragment  
1 μg/ml of DNA 3.08 μM Phosphate
1 μg/ml of a 1 kb DNA fragment 3.08 μM 5'-ends
1 μg of a 1 kb DNA fragment 1.5 pmole = 9.1 x 1011 molecules, 3.0 pmole ends
1 pmole of a 1 kb DNA fragment 0.65 μg
pUC18/19
1 μg of pUC18/19 DNA (2686 bp) 0.57 pmole = 3.4 x 1011 molecules
1 pmole of pUC18/19 DNA 1.77 μ
pBR322 DNA
1 μg pBR322 DNA (4361 bp) 0.35 pmole = 2.1 x 1011 molecules
1 μg of linear pBR322 DNA 0.70 pmole 5'-ends
1 pmole of pBR322 DNA 2.83 μg
1 pmole of 5'-ends of linear pBR322 1.4 μg
M13mp18/19 DNA
1 μg of M13mp18/19 DNA (7249 bp) 0.21 pmole = 1.3 x 1011 molecules
1 pmole of M13mp18/19 DNA 4.78 μ
λ DNA
1 μg of λ DNA (48,502 bp) 0.033 pmole = 1.8 x 1010 molecules
1 pmole of λ DNA 32.01 μ
Molecular Masses of Nucleic Acids
Average molecular mass of a deoxynucleotide base 324.5 g/mole
Average molecular mass of a deoxynucleotide base pair 649.0 g/mole1
Average molecular mass of a ribonucleotide base 340.5 g/mole
1 kb of dsDNA (sodium salt) 6.5 x 105 g/mole
1 kb of ssDNA (sodium salt) 3.3 x 105 g/mole
1 kb of ssRNA (sodium salt) 3.4 x 105 g/mole
λ DNA 3.1 x 107 g/mole2
pBR322 DNA 2.8 x 106 g/mole2
E.coli DNA 3.1 x 109 g/mole2
φX174 DNA 3.6 x 106 g/mole2
1 x 106 g/mole of dsDNA (sodium salt) 1.54 kb
References:
  1. Ausbel. F.Ml, et al., (ed.), Short Protocols in Molecular Biology, Wiley and Sons, Inc. Ny (1999) pp. A2-1
  2. Ausbel. F.M., et al., (ed.), Current Protocols in Molecular Biology, Wiley and Sons, Inc. Ny (1999) pp. A.1B1.
Protein Conversion Data Concentration of Protein to Absorbance of Protein
Protein A280 for 1 mg/ml
IgG 1.35
IgM 1.20
IgA 1.30
Protein A 0.17
Avidin 1.50
Streptavidin 3.40
Bovine Serum Albumin 0.70
Keyhole Limpet Hemocyanin 1.57
Mass of Protein to Mole of Protein
M (g/mole) 1 μg 1 nmol
10,000 100 pmole; 6 x 1013 molecules 10 μg
25,000 40 pmole; 2.4 x 1013 molecules 25 μg
50,000 20 pmole; 1.2 x 1013 molecules 50 μg
75,000 13.3 pmole; 8 x 1012 molecules 75 μg
100,000 10 pmole; 6 x 1012 molecules 100 μg
125,000 8 pmole; 4.8 x 1012 molecules 125 μg
150,000 6.7 pmole; 4 x 1012 molecules 150 μg