Modified Hirt Extract

Lab of Cellular Oncology, NCI

Citation: Uri Arad (1998) Modified Hirt procedure for rapid purification of extrachromosomal DNA from mammalian cells.  Biotechniques24:760 (PMID: 9591124) 

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). That’s more than enough to visualize on GelGreen, GelRed or 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 4ºC 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 ≤ 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.  It’s 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.

•Add 350µl of Buffer III, incubate room temp 10 minutes

•Centrifuge 16,000 x g 10 minutes

•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 5 minutes

•Elute in 75µl.

•Digest the DNA (see notes below) and visualize on an agarose gel precast with GelGreen or GelRed DNA stain (Phenix Research Products).

Notes:  

•Trying to extract more than a million 293 cells using this method can result in contamination with shredded cellular DNA

•Expect SV40 T antigen-replicated target plasmid to be present at >10,000 copies per cell (i.e., 100,000 cells would have about 6 ng of DNA – easily detectable by GelGreen- or GelRed-stained agarose gel). Non-replicated plasmids should be present at ~1000 copies per cell (also detectable by SYBR green)

•In instances where encapsidation efficiency is high, it may be useful to add proteinase-K to liberate the encapsidated DNA. Modify the Biotechniques method by transferring the clarified supernatant to a fresh tube and adding 1 µl of Qiagen proteinase-K stock and incubating the lysate at 37ºC for 10 minutes. Addition of DTT (10mM) may help facilitate capsid digestion.

•If the DNA has been extracted from transfected 293TT cells, it is important to digest with Dpn-I.  This will degrade bacterially-derived plasmids that may still be stuck in the endosomes of transfected cells.  Plasmids that have been replicated by T antigen will lack the bacterial methyl-A modifications that Dpn-I requires for cuting and will therefore be spared from digestion.

•The purified DNA can be polished by removing 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 70º C.  

•The newer dyes GelGreen and GelRed are at least as sensitive as SYBR Green.  In contrast to SYBR Green, GelGreen and GelRed are amenable to being precast into the agarose gel, saving the time and effort of post-staining the gel.

•Some fluorescent DNA dyes (for example, SYBR Green) stain supercoiled DNA relatively poorly, so digestion with a restriction enzyme is desirable.  Choose an enzyme that’s not sensitive to CpG methylation (e.g., BsrGI or MfeI). Nicking enzymes such as Nt.BstNBI (NEB) can also be useful for relaxing plasmid DNA.  My general impression is that GelGreen and GelRed are less sensitive to supecoiling status than SYBR Green.