Preparation of Solutions

Page 1

Preparation of 8% DNA Sequencing Gel

Components Cat. No Amount to use
20% Acrylamide
 Acrylamide BPE170       96,5g  
 Bis-acrylamide    BPE71    3,35g  
 Urea    BPE169    233,5g  
 5X TBE    —    100mL 
 H2O    —    to 500mL  
Urea Mix
 Urea    BPE169    233,5g  
 5X TBE    —    100mL 
 H2O    —    to 500mL  
5X TBE
 Tris Base    BPE152    54g  
 Boric acid    BPE168    27,5g  
 0,5M EDTA (pH 8,0)    BPE120    20mL 
 H2O —       to 1L  
To make an 8% sequencing gel, mix the following in a small flask:
 20% Acrylamide mix    20mL    
 Urea mix    30mL    
 10% ammonium persulfate    0,4mL    
 (freshly dissolved in water)       

For the solution into the barrel of a 50mL syringe and add 50μL of TEMED (BPE150). Mix
rapidly and inject the contents of the syringe (no needle should be used) into a preformed
sequencing gel mould.

Note: Convenient, ready made Fisher BioReagent solutions for key components are also
available. See also Tris-glycine, TBE buffers, acrylamide solutions and water.

Preparation of Tris•CI [Tris(hydroxymethyl)aminomethane] Stock Solutions (1)

Method A
  1. Dissolve 121g Tris Base in 800mL H2O
  2. Adjust solution to desired pH with concentrated HCl
  3. Mix and add H2O to 1L.

Method B
  1. Prepare a 0.1M Tris Base solution: add H20 to 12.1g Tris Base to a total volume of 1L
  2. From the chart below, obtain the volume of 0.1M HCl needed to produce the desired pH, and add to 100mL of 0.1M Tris Base
  3. Mix well
 pH,    0,1M    pH,    0,1M    pH,    0,1M  
 25°C    HCl    25°C    HCl    25°C    HCl  
7,2 89,4mL   7,8 69,0mL   8,4 34,4mL  
7,3 86,8 7,9 64 8,5 29,4
7,4 84 8 58,4 8,6 24,8
7,5 80,6 8,1 52,4 8,7 20,6
7,6 77 8,2 45,8 8,8 17
7,7 73,2 8,3 39,8 8,9 14

Note: The pH of Tris buffers changes significantly with temperature, decreasing approximately 0.028 pH units per 1°C. Tris-buffered solutions should be adjusted to the desired pH at the temperature at which they will be used. Since the pKa of Tris is 8.08, Tris should not be used as a buffer below pH~7.2 or above pH~9.0.

(1) Convenient, ready made Fisher BioReagent solutions are also available.

See also Tris TBE, SSC/SSPE and water

Preparation of Polyacrylamide Stacking and Separating Gels (SDS-PAGE)

 Separating gel (Total volume 15 mL)(1)                      
 Final % acrylamide in gel(2)   5 6 7 7,5 8 9 10 12 13 15
 Stock solutions(3)                                
 30% Acrylamide/0,8% Bis-acrylamide   2,50mL   3,00mL   3,50mL   3,75mL   4,00mL   4,50mL   5,00mL   6,00mL   6,50mL   7,50mL  
 4X Tris Cl, pH 8,8   3,75 3,75 3,75 3,75 3,75 3,75 3,75 3,75 3,75 3,75
 H2O(4) 10% SDS   8,60 0,15   8,10 0,15   7,60 0,15   7,35 0,15   7,10 0,15   6,60 0,15   6,10 0,15   5,10 0,15   4,60 0,15    3,60 0,15  
 10% Ammonium persulfate(5)   0,05 0,05 0,05 0,05 0,05 0,05 0,05 0,05 0,05 0,05
 TEMED   0,01 0,01 0,01 0,01 0,01 0,01 0,01 0,01 0,01 0,01
Page 2

Properties of Aqueous CsCI Solutions @ 20°C

Concentration Percent (w/w) Concentration Percent (w/v) Density (g/mL) Refractive index @ 20°C
20 23,51 1,1756 1,3507
22 26,33 1,1967 1,3528
24 29,24 1,2185 1,3550
26 32,27 1,2411 1,3572
28 35,40 1,2644 1,3594
30 38,66 1,2885 1,3617
32 42,03 1,3135 1,3641
34 45,54 1,3393 1,3666
36 49,18 1,3661 1,3691
38 52,96 1,3938 1,3717
40 56,90 1,4226 1,3744
42 61,00 1,4525 1,3771
44 65,27 1,4835 1,3800
46 69,73 1,5158 1,3829
48 74,37 1,5495 1,3860
50 79,23 1,5846 1,3892
52 84,30 1,6212 1,3925
54 89,62 1,6596 1,3960
56 95,19 1,6999 1,3996
58 101,05 1,7422 1,4035
60 107,21 1,7868 1,4076
62 113,71 1,8340 1,4120
64 120,59 1,8842 1,4167

Southern Blotting Stock Solutions

Prehybridisation Solution (use 0.2mL/cm2 of membrane)
  • 6X SSC (BPE1325)
  • 0.5% SDS (BPE1311)
  • 5X Denhardt's Reagent (BPE515 or see below)
  • 100μg/mL denatured salmon sperm DNA
Hybridisation Solution (use 50μL/cm2 of membrane)
  • 6X SSC (BPE1325)
  • 0.5% SDS (BPE1311)
  • 5X Denhardt's Reagent (BPE515 or see below)
  • 100μg/mL denatured salmon sperm DNA
  • 0.1M EDTA
  • Radioactive or non-radioactive probe DNA

Electrophoresis Stains and Tracking Dyes

Stain/Dye Use
Protein stains
Silver stain Most sensitive protein stain available
Coomassie* Brilliant Blue R-250 General protein stain; more sensitive than Coomassie G-250
Coomassie* Brilliant Blue G-250 General protein stain
Alcian Blue Glycoprotein stain
Fast Green FCF Protein stain for collagenous tissues
Crocein Scarlet Stain used in immunoelectrophoresis
Oil Red O Lipoprotein stain
Light Green SF, Yellowish Protein stain for collagenous tissues
Ponceau S General protein stain
Nucleic acid stains
Methylene Blue Stain for RNA or RNase
Toluidine Blue O Stain for RNA or RNase (alternative to Methylene Blue)
Ethidium Bromide General nucleic acid stain
Silver Stain Very sensitive nucleic acid stain for polyacrylamide gels
Acridine Orange Nucleic acid stain
General stains (Proteins and nucleic acids)
Amido Black 10B General stain, especially suited for nitrocellulose
Basic Fuchsin Glycoprotein and nucleic acid stain
Stains-all General protein and nucleic acid stain
Tracking dyes  
Bromophenol Blue Neutral and basic tracking dye, especially used for SDS–PAGE and DNA sequencing
Bromocresol Green Tracking dye for DNA agarose electrophoresis
Pyronin Y Acidic tracking dye for RNA electrophoresis
Methyl Green Neutral and acidic tracking dye for native DNA
Methyl Red Acidic tracking dye, especially used for isoelectric focusing
Xylene Cyanole FF Tracking dye for DNA sequencing

Haploid DNA Content of Various Organisms

  Size of DNA Weight of DNA
Organism (bp) (Daltons)
Mammals ~3,0 x 109 ~1,9 x 1012
Drosophila ~1,2 x 108 ~7,7 x 1010
Yeast (S. cerevisiae) ~1,6 x 107 ~1,0 x 1010
E. coli ~4,0 x 106 ~2,5 x 109
Bacteriophage T2 ~2,0 x 105 ~1,3 x 108
Bacteriophage ë 48,514 3,1 x 107
pBR322 4363 2,8 x 106
pUC18/pUC19 2686 1,7 x 106

From Molecular Cloning: A Laboratory Manual, second edition (1989).

Page 3

Concentration of DNA in Solution

 Double-stranded DNA (50ìg/mL)    Molecules/mL    Moles/mL    Molar concentration    Molar concentration of termini  
 Bacteriophage   9,78 x 1011  1,62 x 10-12  1,62nM    3,24nM  
 pBR322    1,09 x 1013  1,81 x 10-11  18,1nM    36,2nM  
 pUC18/pUC19    1,77 x 1013  2,94 x 10-11   29,4nM    58,8nM  
 Segment of DNA (1kb)    4,74 x 1013  7,87 x 10-11   78,7nM    157,4nM  
 Octameric double-stranded linker    5,92 x 1015  9,83 x 10-9   9,83µM    19,7µM  

From Molecular Cloning: A Laboratory Manual, second edition (1989).

Preparation of 0.1M Potassium Phosphate Buffer @ 25°C

 Desired pH    Volume of 1M K2HPO4 (mL)    Volume of 1M KH2PO4 (mL)  
5,8 8,5 91,5
6,0 13,2 86,8
6,2 19,2 80,8
6,4 27,8 72,2
6,6 38,1 61,9
6,8 49,7 50,3
7,0 61,5 38,5
7,2 71,7 28,3
7,4 80,2 19,8
7,6 86,6 13,4
7,8 90,8 9,2
8,0 94,0 6,0

Preparation of 0.1M Sodium Phosphate Buffer @ 25°C

 Desired pH    Volume of 1M K2HPO4 (mL)    Volume of 1M KH2PO4 (mL)  
 5.8    7.9    92.1  
 6.0    12.0    88.0  
 6.2    17.8    82.2  
 6.4    25.5    74.5  
 6.6    35.2    64.8  
 6.8    46.3    53.7  
 7.0    57.7    42.3  
 7.2    68.4    31.6  
 7.4    77.4    22.6  
 7.6    84.5    15.5  
 7.8    89.6    10.4  
 8.0    93.2    6.8  

Antibiotic Solutions

   Stock Solution (1) Working concentration
 Concentration  temperature  Storage plasmids   For stringent plasmids    For relaxed  
 Ampicillin    50mg/mL in H2O    –20°C    20µg/mL    60µg/mL  
 Carbenicillin    50mg/mL in H2O    –20°C    20µg/mL    60µg/mL  
 Chloramphenicol    34mg/mL in ethanol    –20°C    25µg/mL    170µg/mL  
 Kanamycin    10mg/mL in H2O    –20°C    10µg/mL    50µg/mL  
 Tetracycline (2)    5mg/mL in ethanol    –20°C    10µg/mL    50µg/mL  

(1) Stock solutions of antibiotics dissolved in H2O should be sterilised by filtration through a 0.22μm filter. Antibiotics dissolved in ethanol need not be sterilised. Store solutions in light-tight containers.
(2) Magnesium ions are antagonists of tetracycline. Use media without magnesium salts (eg, LB medium) for selection of bacteria resistant to tetracycline.

From Molecular Cloning: A Laboratory Manual, second edition (1989).

Page 4

Double-stranded DNA

Density (P) = (0.998)[G + C] + 1.660g/cm3 mole

Plasmid DNA 50% G + C Density
RFI (supercoiled) ds DNA 1,709g/mL
RFII (Nicked) ds DNA 1,54g/mL
  ss DNA 1,726g/mL
  ss RNA 1,90g/mL
Notes

• Nicked DNA binds more Ethidium bromide than does supercoiled DNA
• Ethidium bromide at saturation decreases density of dsDNA ~0.15g/mL
• More Ethidium bromide bound = greater reduction in density
• By comparison, protein has a density of 1.33g/mL

Reference

Schildkraut, C.L., Marmur, J., and Doty, P. (1962) J. Mol. Bio. 4:430-433.

Effective Range of Separation of DNAs in Agarose gels

Amount of Agarose in gel
(%[w/v])		 Effective range of separation of linear DNA molecules (kb)
0,3 5 to 60
0,6 1 to 20
0,7 0,8 to 10
0,9 0,5 to 7
1,2 0,4 to 6
1,5 0,2 to 3
2,0 0,1 to 2

From Molecular Cloning: A Laboratory Manual, second edition (1989).

Effective Range of Separation of DNAs in Polyacrylamide gels

Acrylamide
(%[w/v]) (1) 		Effective range of separation (bp) Xylene Cyanol FF2 Bromophenol Blue (2)
3,5 1,000 to 2,000 460 100
5 80 to 500 260 65
8 60 to 400 160 45
12 40 to 200 70 20
15 25 to 150 60 15
20 6 to 100 45 12

(1) Bis-acrylamide is included at 1/30th the concentration of acrylamide
(2) Numbers are the approximate sizes of fragments of double-stranded DNA with which the dye co-migrates

From Molecular Cloning: A Laboratory Manual, second edition (1989).

Electrophoretic Blotting Buffer

Component Catalogue No Amount to use Final conc.
Tris base BPE152 14,5g 20mM
Glycine BPE381 67,0g 150mM
H2O — 4L  
Methanol BPE1105 1,200mL Adjust pH to 8,0 20%
H2O   6L  

From Molecular Cloning: A Laboratory Manual, second edition (1989).

Migration Rates of Marker Dyes Through Denaturing Polyacrylamide Gels

% Polyacrylamide Xylene Cyanol FF1 Bromophenol Blue1
5 130 35
6 106 26
8 76 19
10 55 12
20 28 8

(1) Numbers are the approximate sizes of DNA (in nucleotides) with which the marker dye co-migrates.

From Molecular Cloning: A Laboratory Manual, second edition (1989).

Relative Migration of Differen Forms of Plasmid DNA on Tris-Acetate Agarose Gels (1)

(1) The exact migration can be affected by agarose gel percentage, electrophoresis time, concentration of Ethidium Bromide, and the size and degree of supercoiling of the DNA
(2) Ethidium Bromide reduces the rate of migration of all plasmid forms. The position of covalently closed circular DNA changes relative to the other forms in the presence of Ethidium Bromide
(3) See pages 232 - 235 for Fisher BioReagents' DNA MW markers
(4) See pages 254 for Fisher BioReagents' Ethidium Bromide

From Molecular Cloning: A Laboratory Manual, second edition (1989)