Reagent and Buffer Recipes

General calculation resources

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

50x TAE

242 g	Tris base
57.1 ml	Glacial Acetic Acid
18.6 g	EDTA
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.

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. 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 serological pipettes to measure out the Acetic acid (use 1 50 ml and 1 10 ml).

5x TBE Tris•Boric Acid•EDTA

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, so measure 100ml of 5x solution in a 1L graduated cylinder and add ddH₂O up to 500 ml to make it 1x. Note: Used as a buffer for Polyacrylamide Gel Electrophoresis.

20x SB Agarose Gel Buffer (Sodium Borate)

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.

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

Dissolve 186.1 g NaEDTA 2 dH20 in 700 ml ddH20

Adjust pH to 8.0 with 10 M NaOH (~ 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

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.