Modified
Hirt Extract
from Uri
Arad (1998) Biotechniques 24:760
Purpose: extract low molecular weight DNA (e.g.,
plasmids) from cultured cells.
This method uses neutral pH for lysing the cells (as opposed to the
alkaline conditions in the traditional Hirt extract). Non-supercoiled plasmid species are recovered more
efficiently in this method compared to other methods. 293TT cells transfected with SV40 Ori+ plasmids
can yield more than 10,000 copies of plasmid per cell by day 2 (~50 ng per
million cells). ThatÕs more than
enough to visualize on SYBR Green-I stained agarose gels.
Buffer I
(resuspension) (25ml)
water
- 23.2
ml
50mM Tris,
pH 7.5 - 1.25 ml of 1M
10mM
EDTA
- 0.5 ml
of 0.5M
50µg/ml
RNase A - 25 µl of 50
mg/ml stock
RNase T1
cocktail - 25 µl of 20
U/µl stock (Ambion)
Buffer II
(lysis) (25ml)
water
-
22 ml
1.2%
SDS
-
3 ml of 10%
Buffer
III (precip) (35ml)
3M CsCl
- 17.68 g
1M Potassium
acetate - 7 ml of 5M (4.91 g/10 ml)
0.67M Acetic
acid - 1.35ml of glacial (17.4M)
QC with
water
water
- 34.4 ml
60%
Ethanol
- 63 ml of 190 proof
10 mM Tris,
pH 7.5
- 1 ml of 1M
50 µM
EDTA
- 10 µl of 0.5M
80 mM
Potassium acetate - 1.6 ml of 5M
Store all
buffers at 4¼C for up to one year.
Re-warm
buffers to room temp prior to use.
100 ml water
10 ml Qiagen
EB buffer
20µl of 0.5M
EDTA
„Prep ² 1
million cells (i.e., confluent ~24-well well).
„Trypsinze cells, resuspend in DMEM/10. Save floaters, if any.
„Spin cells,
aspirate supe, wash 1x with PBS.
„Resuspend
cells in 250µl of Buffer I. ItÕs
also OK to suspend the cells in up to 100µl of plain PBS and add Buffer I to
bring the volume up to 250µl – the extra salt appears not to affect the
extraction. Suspension can be
frozen at -80 indefinitely.
„Thaw, add
250µl of Buffer II, incubate 5 minutes.
„(Optional): It is theoretically possible that
plasmids residing within papillomavirus capsids will not be fully extracted by
the SDS. If this is a concern, it
is acceptable to add 0.1% proteinase K stock (Qiagen) during neutral
lysis. Proteinase K digest 37¼ C
10 min.
„Add 350µl
of Buffer III, incubate room temp 10 minutes
„Centrifuge
16,000 x g 10 minutes (refrigerated, if possible).
„Invert the
tube a few times, centrifuge 10 more minutes.
„Load supe
onto blue Qiagen miniprep columns (#27106).
„Wash column
with 750µl of wash buffer
„Spin column
dry for 10 minutes
„Elute in
75µl.
„(Optional): Digest with Dpn-I. This will digest bacterially-derived
plasmids that may still be stuck in the endosomes of transfected cells. Plasmids that have been replicated by T
antigen will lack bacterially methyl-A modifications that Dpn-I needs to cut
and will therefore be spared from digestion.
„(Optional):
remove linear fragments of cellular DNA (and Dpn-I fragments) by digestion with
Plasmid Safe Exonuclease V (Epicentre).
Both Plasmid Safe and Dpn-I work well in NEB buffer #4. Heat inactivate the Plasmid Safe 30 min
70¼ C.
„Digest
extracted plasmids with diagnostic restriction enzymes.
„Visualize
on an agarose gel stained with SYBR Green-I (Molecular Probes).