Hello Alveena Muti

First and foremost, you need to prepare the stock solution of Tris-HCl pH 8.0, EDTA and SDS.

Preparation of 1 M Tris-HCl (pH 8) (100mL).

Weigh Tris Base 12.11g in 90mL distilled water and add approximately 4.0-4.2 mL HCL to adjust the pH to 8.0. Make up the volume to 100mL.

Preparation of EDTA 0.5 M (pH 8.0) (100mL).

Add 14.8 g EDTA + ~3.0-4.0 g NaOH to adjust the pH (or you may use 18.6 g EDTA-Na.2H2O + ~2.0 g NaOH) in distilled water. Use magnetic stirrer to stir the solution. Make up the volume to 100mL with distilled water. Please note: You need to adjust the pH by NaOH for solubility of EDTA.

Preparation of 10% SDS (stock).

You may prepare 10% SDS solution (100 ml) by adding 10g SDS in 80 ml of deionized water and heat it at 40-50 degree C until it dissolves and subsequently make up the volume to 100 ml.

For proteinase inhibitor use Phenylmethylsulfonylfluoride (PMSF) at 1mM.

Prepare a 100 mM stock solution of PMSF by adding 17.4 mg of PMSF per milliliter of isopropanol. Store in aliquots at −20°C.

To prepare 100mL of 2% SDS lysis buffer, add the following from the above stock solutions.

1. 5ml of 1M Tris-HCL (pH 8.0),

2. 2ml of 0.5M EDTA (pH 8.0),

3. 20ml of 10%SDS stock,

4. 10mL of glycerol,

5. 1ml of (100mM) PMSF should be added to 100ml of 2% SDS lysis buffer fresh at the time of lysis.

Make up the volume to 100mL with distilled water.

Best.

Malcolm Nobre

Thank you so much. It helped alot.

I want to ask one more question, you have prepared this protocol for 1x or 2x buffer?

Regards

Alveena Muti it is for 1X buffer.