Modified Hirt’s Extraction of low molecular weight DNA and DpnI sensitivity
assay:
1. Adherent cells, remove culture media by aspirating it off from the dish.
Suspension cells, pellet down the cells and remove the media.
2. Wash the cells with PBS (adherent cells in plate and suspension cells by
centrifugation).
3. Add 2.4ml of solution A in plate and incubate at RT for 10min (for suspension
cells resuspend them and then add solution A)
4. Scrap off the lysed cells from plate (adherent cells) and transfer that into 15ml
tube.
5. Add 1.2 ml of solution B. mix the tube content gently and incubate at ice for
10min.
6. Centrifuge to remove cell debris at 8000rpm for 10min at 4.0oC.
7. Transfer the supernatant into fresh 15ml tube and phenolize by adding 2ml
phenol:CHCl3:IAA mixture. Vortex the tube for 30sec and centrifuge again at
8000rpm for 10min.
8. Transfer aqueous phase into fresh 15ml tube and then add 0.6 volume of Isopropanol (2-propanol), mix it by inverting the tube couple of times.
9. Incubate at RT for 15min.
10. Centrifuge to pellet the DNA at 8000rpm for 10min at RT.
11. Decant the supernatant carefully; dry the pellet by leaving them at RT for 2030min.
12. Resuspend the pellet in 400ul of TE +RNAase and incubate at 37oC for 30min.
Transfer DNA into 1.7ml eppendorff tube and add proteinase K and SDS
(50ug/ml+0.5%SDS final concentration).
13. Incubate at 50oC for 20-30min followed by phenolization, adding equal volume of
phenol CHCl3:IAA. Wash once more with CHCl3:IAA and then precipitate DNA
using sod. Acetate and Ethenol. (0.3M NaAcetate and 2.5 volume of Ethenol).
14. Centrifuge to pellet the DNA, wash with 70% ethanol followed by drying.
15. Resuspend DNA into 30-50ul (depending the efficiency of elution) of sterile
water and now ready for DpnI sensitivity analysis.
16. For DpnI analyses digest 10% with EcoRI (to linearize, if not TR plasmid digest
with enzyme which cuts only once) and rest 90% with EcoRI and DpnI together
with sufficient amount of enzyme.
17. Digest overnight, run them on 0.8%agarose and transfer to gene screen membrane
by using vacuum transfer or capillary transfer method.
18. After transfer immerse the membrane in 0.4N NaOH for 1min followed by
neutralizing it with Neutralization Soln [0.2M Tris (pH7.5), 1X SSC].
19. Cross-link DNA by baking the membrane at 80oC for 2h under vacuum (do not
use gel drier for cross-linking). Now the membrane is ready for hybridization.
20. Pre-hybridize the membrane using hybridization solution (without probe) at 67oC
for at-least 1h.
21. Remove prehyb. solution and add denatured probe in Hyb. Soln and hybridize at
67oC for at-least 6h.
22. Wash the membrane with Wash I (2XSCC, 1%SDS) for 20min at 67oC followed
by washing it with Wash II (0.2XSSC, 0.1% SDS).
23. Expose the membrane to PhosphoImager plate by putting it in some secure bag so
the content does not leak (the membrane is going to be exposed wet, Do not dry
the membrane).
Solutions
Solution A:
Solution I +Soln II (1:2)
Solution B:
Solution III of mxiprep DNA
TE RNAse: Tris 10mM (pH 8.0) 1mM EDTA (pH8.0) and 1ug/ml RNAse.
SSC and HYB solution
20X SSC (1L)
NaCl
175.4gm
Sod. Citrate 88.2gm
Hybridization Soln.
2XSSC
0.5% Blocking Reagent
5.0% dextran Sulphate
0.1%SDS