Protocols

Here is the first protocol I learned (adapted from Sigma Manual)

Sigma Mini Prep protocol:

Before beginning:

-          Make sure Wash solution has ethanol added

-          Add RNase to Resuspension solution

Protocol:

1.       Pellet 1-5ml of overnight culture

2.       Resuspend in 200ul of RNase Resuspension solution

3.       Add 200ul of lysis solution and let sit for up to 5 minutes, mix by inverting not centrifuge

4.       Add 350ul of neutralization solution and mix by inverting the tube

5.       Centrifuge at 13000rpm for 10 minutes

6.       While waiting, prepare column by adding 750ul of column prep solution, centrifuge at 13000 for 30-60s and discard flow-through

7.       Remove supernatant and place into column

8.       Centrifuge column for 30-60s (13000rpm)

9.       Discard flow through

10.   Add 750ul of Wash solution and centrifuge for 30-60s at 13000rpm

11.   Discard flow through, Spin again for 30-60s at 13000rpm and discard flow through

12.   Apply 50ul of EB buffer directly to column filter and let stand for 5 minutes

13.   Spin column for 30-60s at 13000rpm

14.   Flow through contains DNA, quantify on nanodrop. 

Pl Please let me know if you like this and I will post more, otherwise I will stop.