Reagent and Buffer Recipes

General calculation resources

• Sigma – Mass Molarity Calculator
• Sigma – Solution Dilution Calculator

Gel Electrophoresis

50× TAE (Tris•Acetate•EDTA) Agarose Gel Electrophoresis Buffer

Used as a buffer for Agarose Gel Electrophoresis.

242 g	Tris base
57.1 mL	Glacial acetic acid
100 mL	0.5 M EDTA (pH 8.0)
to 1 L	ddH20

Prepare by filling bottle with 700 ml of ddH₂0 and adding the above chemicals. Adjust volume to 1 L with ddH₂0. The final solution should have a pH of ~8.5.

Unused or leftover acetic acid must be disposed of in a chemical waste bottle located in the fume hoods. To avoid having leftover Acetic acid use a serological pipette to measure out the glacial acetic acid.

Working concentration is 1×, so measure 400 ml of 50× solution in graduated cylinder and then pour into 20 L carboy and fill to 20 L with ddH₂0; if filling a 10 L carboy use 200 ml of stock.

5× TBE Tris•Borate•EDTA Polyacrylamide Gel Electrophoresis

Used as a buffer for Polyacrylamide Gel Electrophoresis.

54 g	Tris base
27.5g	Boric Acid
20 mL	0.5 M EDTA (pH 8.0)
to 1 L	ddH20

Working concentration is 1× (for standard gels) or sometimes 0.5× (for PFGE). For 1×, measure 100ml of 5x solution into a 1L graduated cylinder and add ddH₂O up to 500 ml to make it 1× or up to 1000 ml to make it 0.5×.

6× Bromophenol Blue Agarose Gel Loading Buffer

24 ml	Glycerol
0.015 g	Bromophenol Blue
2.4 ml	50× TAE loading buffer
to 40 ml	ddH20

The final concentrations at 6× are 60% glycerol, 0.25% bromophenol blue, 3×TAE. At 1× are 10% glycerol, 0.0417% bromophenol blue, 0.5× TAE.

Note: Generally, your samples will be in PCR buffer that adds extra salt and buffering capacity. If you are adding pure DNA to this loading buffer, you may need to add PCR buffer to achieve consistent band mobilities.

Agarose Gel DNA Ladder Working Solution

To prepare the Invitrogen 1 Kb Plus ladder for loading on gels.

Create a 1:6 dilution of ladder combining it with Loading Buffer so that it reaches a 1× final concentration.

166.6 ul	Ladder
166.6 ul	6× Loading Buffer (above)
666.8 ul	Milipure H2O

If you load 6 µl of this Working Solution, it will contain 0.5 µg of ladder and this image from the ThermoFisher website shows the number of ng of DNA in each band on the gel. You can compare the density of your bands to those of the ladded to estimate DNA concentrations.

Stock solutions

Tris-HCl, 1 M

121 g Tris base in 800 ml ddH20

Adjust to pH 8.0 with HCl

Mix and add ddH20 to 1 L

EDTA, 0.5 M (pH 8.0)

Dissolve 186.1 g Na•EDTA•2H20 (EDTA disodium salt dihydrate) in 700 ml ddH20

Adjust pH to 8.0 with 10 M NaOH ( It should take ~ 50ml)

Mix and add ddH20 to 1 L

NaOH, 10 M

Dissolve 400g NaOH in 450 ml ddH20.

Mix and add ddH20 to 1 L

*Warning: Add NaOH pellets slowly! Dissolving is very, very exothermic.

Potassium acetate, 5 M

29.5 ml glacial acetic acid

KOH pellets to pH 4.8 (several)

ddH20 to 100 ml

Store at room temperature

NaCl, 1M

Dissolve 58.4 g of NaCl in 800 ml ddH20

Mix and add ddH20 to 1 L

CaCl₂, 1 M

Dissolve 110.9 g of CaCl2 in 800 ml ddH20

Mix and add ddH20 to 1 L

Aliquot into 2 500 ml bottles and autoclave

MgSO₄, 1 M

Dissolve 120.3 g of MgSO4 in 800 ml ddH20

Mix and add ddH20 to 1 L

Aliquot into 2 500 ml bottles and autoclave

RNase A, 5 mg/ml

Dissolve 100 mg of RNase A in 20 ml of 0.05% glacial acetic acid, and transfer to a 50-ml conical tube

Place the tube in a boiling-water bath for 15 minutes

Cool the solution, and neutralize by adding 120 μl of 1 M Tris (pH 8.0)

Distribute 1 ml aliquote into 1.5 ml MFT, and store at -20 C

rNTP, 100 mM

For in vitro transcription or deoxyribozyme reactions:

1. Dissolve 1 g of desired NTP in 10 ml ddH₂O:
   • ATP - Adenosine 5′-triphosphate disodium salt (MW 551.14)
   • GTP - Guanosine 5′-triphosphate sodium salt hydrate (MW 523.18)
2. pH to 8.0 with 1 M NaOH. It takes approximately 1.0–1.5 ml.
3. Add ddH₂O to final volume of:
   • GTP - 18.14 ml
   • GTP - 19.11 ml
4. Store at –20°C in 1.0–1.5 ml aliquots.

HEPES•NaOH, 1M, pH 7.0

pH buffer with less temperature dependence than Tris.

To make 100 ml:

1. Dissolve 23.83 g HEPES (Free Acid) in 80 ml ddH₂O.
2. pH to 7.0 with 6 M NaOH. It takes approximately 2.0 ml.
3. Add ddH₂O to final volume of 100 ml.