Method for seperation of plasmid dna from large bacterial chromosomal dna
Answers
please following this protocol step.
Materials Required:
Lysis buffer (40mM tris-acetate PH 7.8, 20mM sodium-acetate, 1mM EDTA, 1% SDS), chloroform, 5 MNacl, 70% EtOH, TE buffer.
Protocol:
1- Harvest bacteria colony into 1.5 ml Eppendorf tube.
2- resuspend cell pellet in 200 µl of lysis buffer (40 mM Tris-acetate PH 7.8, 20µl sodium – acetate, 1mM EDTA, 1% SDS) by vigorous pipetting.
3- Add 66 µl of 5M NaCl solution (to remove most protein and cell debris).
4- Centrifuge at 12000 rpm for 10 min at 4℃.
5- Transfer the clear supernatant into new vial, add equal volume of chloroform.
6- Shake tube gently at least 50 times until a milky solution completely formed.
7- Centrifuge 12000 rpm for 3min.
8- Transfer the supernatant to another vial
9- Add equal volume of cold absolute ethanol to precipitate DNA.
10-wash DNA with 70% Ethanol
11-dry DNA and dissolve in 50µl TE buffer or double distil water.
The yield of DNA around 50-150ug per 109 cells