Stock Solutions required to prepare Lysis solutions:

1N NaOH – 1L

40g NaOH pellets

Adjust volume to 1000ml with ddH2O

OR

100ml 10N NaOH QS to 1L with sterile H2O

 

10N NaOH - 0.5L

200g NaOH pellets

Adjust volume to 500ml with ddH2O

 

1M Tris-HCl pH 7.6 – 1L

121.14g Tris base m.w. 121.14

Adjust pH to 7.6 with concentrated HCL

QS to 1L with ddH2O

Autoclave

 

0.5M EDTA pH 8.0 – 1L

186.12g Disodium-EDTA·2 H2O (m.w. 372.24g)

(**Verify that you are using the disodium salt, not the free acid – check the Mol. Wt.)

Adjust pH to 8.0 with 10 N NaOH

Heat a 500ml bottle of sterile H2O 4 minutes in the microwave. Pour 500ml into a beaker with a stir bar and begin stirring. Slowly add EDTA to dissolve and begin adjusting the pH with 10N NaOH. (EDTA dissolves slowly and will not dissolve completely until it has reached the proper pH, take it slowly with this solution.)

QS to 1L with ddH2O

Autoclave

 

20% SDS – 1L

200g SDS

Heat a 500ml bottle of sterile H2O 4 minutes in the microwave. Pour all of it into a 1L graduated cylinder and slowly add SDS (allow SDS to dissolve between additions). Be careful not to let the solution suds up with stirring, the addition of the SDS or additional water. QS to 1L with sterile H2O.

Lysis soulutions: (Note: Assign stock solutions log #’s and record in Stock Solutions & Buffers Notebook.)

SDS/NaOH (final cc 1%/02.N) – 1L

200ml of 1N NaOH

50ml of 20% SDS

Add 750ml sterile dd H2O

Be careful with additions so that SDS does not suds up and affect final volume.

 

TE/RnaseA (final cc 50mM/10mM)– 1 L

Add 50ml of 1M Tris HCL pH 7.6

Add 20ml of 0.5M EDTA pH 8.0

Add 925ml sterile ddH2O

Just before use add 0.5ml of 10mg/ml Rnase A to 100ml of TE buffer.

 

3M KOAc pH 5.0 – 1L

292.42g Potassium Acetate

Adjust pH to 5.0 with glacial Acetic Acid

Heat 500ml bottle of sterile water 4 minutes in the microwave, while heating water weigh 292.42g KOAC into a medium beaker. Poor approximately 400ml of the hot water into a 2L graduated cylinder with a stir bar and begin stirring. Gradually add KOAc to the water to dissolve. Rinse beaker with minimal hot H2O. Adjust pH with glacial Acetic Acid to approximately pH 5.8 (volume should be around 800ml). Than slowly continue adjusting pH to 5.0 (allowing equilibration between additions). Water addition will lower the pH a little (enough to bring it below 5.0, so be very careful as you get to 1L final volume.)