Reagent and Buffer Recipes

General calculation resources

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

50x TAE Tris•Acetate•EDTA

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.

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

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

5x TBE Tris•Borate•EDTA

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

20x SB Agarose Gel Buffer (Sodium Borate)

An alternative buffer for Agarose Gel Electrophoresis.

38.2 g	Borax
10 g	Boric Acid
to 1 L	ddH20

Mix with a stir bar until everything is dissolved and liquid is clear. Pour into one of the 20 L carboys and fill to 20 L with ddH₂0 to make it 1x.

6x Bromophenol Blue Gel Loading Buffer

30 ml	Glycerol
0.01 g	Bromophenol Blue
to 50 ml	ddH20

DNA Ladder with Loading Dye

375 ul	Loading Buffer (above)
250 ul	Ladder
325 ul	10x PCR Buffer
3 ml	ddH20

Makes ~4ml. Aliquot to 1.5 ml centrifuge tubes.

To prepare the Invitrogen 1kb+ ladder: Create a 1:10 dilution of ladder in water, accounting for the addition of your dye of choice (see example below)

100 ul	Ladder
166.6 ul	6x Loading Buffer (above)
733.4 ul	Milipure H2O

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.