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

 

Column Wash (100 ml)

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.

 

Elute (100ml)

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).