Freeze thaw lysis of E.coli

Freeze thaw lysis of E.coli

Buffer A-   50mM Tris pH8, 150mm NaCl, 5mM EDTA, PMSF 1mM

                                                       Or     

                                            1xPBS, 1mM PMSF

1.      Pellet down cultures (100mg)

2.      Resuspend cells in 200ul of buffer A

3.      Add 20ul of lysozyme (stock 10mg/ml)

4.      Incubate tubes at 30ᴼC shaking for 1hr

5.      Freeze the cells in liquid nitrogen

6.      Thaw the cells in 37ᴼC water bath

7.      Repeat steps 5 and 6 for 5-8 cycles.

8.      Spin @ 13000rpm for 20mins

9.      Separate supernatant and pellet

10.  Resuspend pellet in 250ul of 1x SDS PAGE loading buffer

11.  Add 50ul of SDS PAGE loading buffer  to the supernatant

12.  Boil samples 95ᴼC for 10min

13.  Load equal amount on gel