Appendix 4: Buffer formulations
- COMMONLY-USED STOCK SOLUTIONS
The following stock solutions are commonly used in the preparation of many reagents used in molecular biological procedures. Remember to use aseptic technique with sterile stocks. Use deionized or glass-distilled water to make reagents. Decrease volumes made as appropriate. It is also useful to maintain a store of steriledeionized water.
ß-mercaptoethanol (BME)
BME is purchased as a liquid at a concentration of 14.4 M. Store at 4 ˚C in a dark bottle. Do not autoclave BME or buffers containing it. Open BME in a hood.
1.0 M Calcium Chloride
Dissolve 14.7 g of CaCl2• 2H2O in 80 mL of deionized water. Adjust the volume to 100 mL with deionized water. This may be stored at room temperature indefinitely.
100 mM Calcium Chloride(for preparation of competent cells)
Add 50.0 mL of 1.0 M CaCl2to 450 mL of deionized water and mix. Sterilize by autoclaving. Store at 4 ˚C.
0.5 M EDTA, pH 8.0
Dissolve 186.1 g of disodium ethylenediaminetetraacetate•2H2O in 800 mL of deionized water that is being mixed on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (about 20 g of pellets). The EDTA will not completely dissolve until the pH has reached 8.0. Add the NaOH slowly so as not to adjust the pH above 8.0. Adjust to 1000 mL with deionized water. Dispense into appropriate containers and sterilize by autoclaving. Store at room temperature.
10 mg/ mL Ethidium Bromide
Dissolve 0.25 g of ethidium bromide in 25.0 mL of deionized water. Stir at room temperature for several hours to ensure that the dye is completely dissolved. Store in a foil-wrapped or brown glass bottle at room temperature.
3.0 M Sodium Acetate (pH 5.2) (anhydrous MW = 82.03)
Add 24.6 g of sodium acetate (anhydrous) to 70 mL of deionized water and mix to dissolve. Adjust the pH to 5.2 with glacial acetic acid. Make to 100 mL with deionized water. Sterilize by autoclaving and store at room temperature.
5.0 M Sodium Chloride
Dissolve 292.2 g of NaCl in 700 mL of deionized water. Heat may be necessary to completely dissolve the NaCl. Adjust to 1000 mL with deionized water and sterilize by autoclaving. Store at room temperature.
20% SDS
Add 200 g of molecular biology grade sodium dodecyl sulfate (often called sodium lauryl sulfate) to 800 mL of deionized water. Dissolve with heating on a hot plate. Adjust the pH to 7.2 with a few drops of 6N HCl. Adjust to 1000 mL with deionized water. Do not autoclave this stock. Store at room temperature.
4.0 N Sodium Hydroxide
Add 160 g of NaOH pellets to 800 mL of deionized water. Dissolve on a stir plate as the solution will get hot from an exothermic reaction. After all pellets are dissolved, allow the solution to cool and adjust to 1000 mL with deionized water. Store at room temperature in a polypropylene bottle (NaOH should not be stored in glass bottles).
1.0 M Tris, pH 8.0
Dissolve 121.1 g of Tris base in 800 mL of deionized water. To avoid inhalation, wear a mask when weighing out the dry powder. Adjust the pH to 8.0 with concentrated HCl (about 42 ml). Cool the solution to room temperature and adjust to 1000 mL with deionized water. Dispense into appropriate containers and sterilize by autoclaving. Store at room temperature.
NOTE: The pH of Tris buffers is temperature-dependent. Make all measurements at room temperature. The pH will decrease approximately 0.03 pH units for each degree Celsius that the temperature increases.
NOTE: Many pH electrodes do not accurately measure the hydrogen ion concentration of Tris buffers. Be sure that the electrode you are using is capable of measuring Tris.
50x TAE
242 g Tris base
57.1 mL glacial acetic acid
100 ml of 0.5 M EDTA (pH 8.0)
- DNA AND ENZYME SOLUTIONS
DNA solutions (plasmids, lambda, etc.) are generally supplied at a known concentration in TE buffer (10 mM Tris, pH 8.0; 1 mM EDTA). Dilutions of DNA stocks should be made into TE buffer. Wear gloves when handling DNA stocks and exercise caution to avoid nuclease contamination. Store DNA solutions at 4 ˚C if you are using them frequently, or at -20 ˚C for long-term storage.
Restriction enzymes, ligase, and many other DNA modifying enzymes are prepared in buffers containing 50% glycerol and must be stored at -20 ˚C. The concentrations of commercially available enzymes varies from 5-20 units/ μL, but it is best not to dilute them for use. Dilution often results in rapid loss of activity and is time-consuming. Addition of an excess amount of enzyme makes reactions proceed faster. Some enzymes are purchased as dry powders and are prepared as follows:
Lysozyme (10 mg/ mL in 10 mM Tris, pH 8.0)
Make lysozyme solution immediately (1-4 hours) before use. To 11.9 mL deionized water add, 0.12 mL of 1.0 M Tris, pH 8.0 and 0.12 g lysozyme. Dissolve the lysozyme and keep the solution on ice.
Proteinase K (10 mg/ ml)
Dissolve 25 mg of proteinase K in 2.5 mL of sterile, deionized water. Dispense into sterile microcentrifuge tubes in 500 μL aliquots and store at -20 ˚C.
RNase A (10 mg/ mL in 10 mM Tris, pH 7.6, 15 mM NaCl)
Add 50.0 μL of 1.0 M Tris, pH 7.6 , 15.0 μL of 5 M NaCl, and 50 mg of RNase A to a total volume of 5.0 mL of deionized water:
Dissolve the RNase A and place in boiling water bath for 10 minutes to inactivated any DNase that is a contaminant. Transfer to sterile microcentrifuge tubes in 500 μL aliquots and store at -20 ˚C.
Gibson cloning recipes
Gibson Master Mix Isothermal Reaction Buffer:
Makes approximately 9 mL. Store in 350 μL aliquots at -20C.
| Component | Amount |
| 1M Tris-HCl pH 7.5 | 3 mL |
| 1M MgCl2 | 300 μL |
| 100 mM dNTP | 60 μL |
| PEG-8000 | 1.5 g |
| NAD | 19.9 mg |
| 1M DTT | 300 μL |
| Water | 2.22 mL |
Gibson Master Mix:
Mix up the reagents well. The solution is rather viscous, so pipette slowly.
Make 100 uL aliquots and store at -20C.
| Component | Amount |
| Isothermal Reaction Buffer | 320 μL |
| T5 exonuclease (10U/μL) | 0.64 μL |
| Phusion DNA Polymerase (2 U/μL) | 20 μL |
| Taq DNA Ligase (40 U/μL) | 160 μL |
| Water | 700 μL |
III. MEDIA
LB (Luria-Bertani) Medium
Add to 1000 mL deionized water:
10.0 g tryptone
5.0 g yeast extract
10.0 g NaCl
Dissolve all ingredients and adjust the pH to 7.2 with NaOH (about 1 mL of 4 N NaOH). Sterilize by autoclaving. Prepare agar plates by adding 15 g/L agar prior to sterilization.
The following sterile stocks should be aseptically added to autoclaved and cooled (to 55 C) media. For cold storage, aliquot stocks into small volumes to avoid repeated freezing and thawing.
100 mg/mL IPTG
Dissolve 1.0 g of isopropylthio-ß-D-galactoside (IPTG) in 8.0 mL of deionized water. Adjust the volume to 10.0 mL with deionized water. Sterilize through a 0.2 µm membrane filter and dispense in 2-3 mL aliquots in sterile vials or tubes. Store at -20 ˚C. Add 1.0 mL per liter ofsterilizedmedium that has cooled to 55 ˚C.
NOTE: IPTG can be left out of media used for alpha-complementation if pUC or pGEM-type vectors are being used. These are such high copy number vectors that there is enough expression of the a-fragment of ß-galactosidase to obtain activity (i.e.blue colonies).
40 mg/mL X-gal
Dissolve 0.4 g of 5-bromo-4-chloro-3-indoyl-ß-D-galactoside (X-gal) in 10 mL of dimethlyformamide (DMF) in a glass or polypropylene tube. DMF WILL DISSOLVE POLYCARBONATE. Store in 2-3 mL aliquots in foil-wrapped vials at -20 ˚C. Do not sterilize. Add 1.0 mL per liter of sterilizedmedium that has cooled to 55 ˚C, giving a final concentration of 40 µg/mL.
50 mg/ mL Kanamycin
Dissolve 0.5 g of Kanamycin (sodium salt) in 10 mL of deionized water. Sterilize through a 0.2 µm membrane filter. Dispense in 2-3 mL aliquots in sterile 1-mL vials or sterile tubes. Store at -20 ˚C. Add 1.0 mL per liter of sterilizedmedium that has cooled to 55 ˚C, giving a final concentration of 50 µg/ mL. Adjust the volume added accordingly for different concentrations.
Kanamycin is stable for about a month as a stock or in agar plates stored in the refrigerator.
Buffers Needed for SDS-PAGE
For the SDS-polyacrylamide gels you poured, we provided the following reagents:
- 10% SDS Solution: 10 g of SDS dissolved in a final volume of 100 mL of deionized water;
- 40% Acrylamide Solution: This solution is 37.5:1 Acrylamide: Bis-acrylamide.
- 10% Ammonium Persulfate Solution: 0.1 g of ammonium persulfate diluted to a final volume of 1 mL with deionized water;
- TEMED: use as a neat solution;
- 4x Resolving Gel Buffer: Combine 45.42 g Tris Base and1 g SDS then add deionized water to a final volume that is almost 250 mL ,carefully adjust the pH to 8.8 with HCl, and then add enough water to bring the final volume to 250 mL.
- 4x Stacking Gel Buffer: Combine 15.14 g Tris Base and 1g SDS then add deionized water to a final volume that is almost 250 mL, carefully adjust the pH to 6.8 with HCl, and then add enough water to bring the final volume to 250mL.
- 4x SDS sample buffer: For 10 mL: Combine 2 mL 1M Tris pH 8, 0.8g SDS, 4 mL 100% glycerol, 1 mL 0.5M EDTA, and 8 mg of Bromophenol Blue and then add deionized water to final volume. Add a denaturing agent if required, such as 5% BME.
- 5X Laemmli Running Buffer:Combine 152 g Tris base, 720 g glycine, and 50 g SDS add water to a final volume of 10L. This buffer should be pH 8.5, and normally should not be adjusted;
- Staining solution: Combine 450 mL dH2O, 450 mL methanol and 100 mL glacial acetic acid. Add 1.0 g of Coomassie Blue R250. The stain can be re-used a few times but the gel may need to stay in used stain a little longer than in fresh.
- Destaining solution: Combine 875 mL dH2O, 50 mL methanol and 75 mL glacial acetic.
Buffers used for the Ni column (Lab 7)
Resuspension buffer:
50 mM NaH2PO4
300 mM NaCl
Adjust pH to 8.0 with NaOH
You are responsible for deciding on appropriate equilibration, wash and elution buffers for use on the column, so those recipes are not provided here.
Protein Buffer:
50 mM Tris, pH 7.8
0.1 M K2SO4