DNA extraction technique for biopsy tissue

Adapted by C.S. Baker (with further modifications by C. Conway, 11/10/99)

 

Reagents:

            Lysis buffer, pH 8.0                                                 To make 500 ml lysis buffer:

                        50  mM  Tris HCl, pH 8.0                                        250 ml 0.1 M  Tris base

                        200 mM NaCl                                                                                   (Sigma T-1503)

                        20 mM EDTA, pH 8.0                                              146 ml 0.1 N  HCl

                        1% SDS                                                                      20 ml 5 M  NaCl

                                                                                                            20 ml 0.5 M  EDTA

            Proteinase K:  10 mg/ml                                                                pH 8.0

            7.5 M ammonium acetate                                                   50 ml 10%  SDS

            Phenol, equilibrated, pH 8.0                                                          14 ml ddH2O

            Chloroform-isoamyl alcohol (24:1)                                              

            95% ethanol

            70% ethanol

           

 

Note:  all instruments and reagents should be sterile.  Scalpels, forceps and other instruments should be cleaned with 70% EtOH and flamed over burner between samples.

 

1)  Remove small section (~20mg) of sample.  Wash with ddH2O.  Dice into small pieces on glass or plastic plate covered with parafilm.  Change parafilm between samples.

 

2)  Place diced sample in 2.0 ml microcentrifuge tube containing 500 ul lysis buffer.  Bring total volume to 900 ul with lysis buffer.

 

3)  Add 100 ul proteinase K (10mg/ml) and incubate overnight  at 55o C with gentle rotation.

 

4)  Add equal volume of equilibrated phenol.  Vortex briefly.  Rotate or rock at room temp for 10-15 min.  Spin down 15-20 min at 13,000 rpms.  Carefully draw off supernatant and place in new 2.0 ml tube.

 

5)  Add equal volume of chloroform-isoamyl alcohol (24:1).  Vortex briefly.  (Can leave sample in frig overnight at this stage, if need to stop).  Rotate or rock at room temp for 10 min.  Spin for 15 min at 13,000 rpms.  Draw off supernatant and place in new 2.0 ml tube. 

 

6)  Repeat step 5.

 

7)    Split into 2 tubes.  Add ˝ volume ammonium acetate (7.5 M).

 

8)  Add 2 ˝ volumes (compared to volume before ammonium acetate added) of cold 95% EtOH.  Place in freezer.

 

 

 

9)  DNA may spool into white cottony ball (high quality DNA) after about an hour or less at minus 20o C (if doesn’t, still may have good DNA).  Remove spooled DNA with sterile 1000 ul pipettor.  Place in new tube with 200 ul 70% EtOH to wash.  Spin down for 10 min at 7,000 rpms to get pellet.  Pour off or pipette off EtOH (be careful not to lose pellet). Place tube in block or incubator for 10 min at ~55o C to dry off remaining EtOH.  (Can leave dried DNA at room temp at this stage if need to stop for awhile.)  Dissolve in 150 ul TE at 55o C for 1 hr.  (After pipetting TE into tube, use pipettor tip to scrape DNA off bottom of tube, and pump solution several times to mix). Label with “H” (for high quality).

 

10)  Remaining DNA in 2 tubes (after spools pulled off) can be collected by spinning down to pellets at 7000 rpms for 20 min. (if no pellet seen, may still have DNA at bottom of tube).  Pour off 95% EtOH.  Add ~150 ul 70% EtOH to wash pellet.  Spin again at 7000 rpms for 10 min.  Pour off EtOH.  Dry for 10 min at ~55o C.  Dissolve in 25 ul TE (each tube) at 55o C for 1 hour. (After pipetting TE into tube, use pipettor tip to scrape DNA off bottom of tube, and pump solution several times to mix).

 

11)  Quantify DNA concentration in each tube.  Standardize concentrations of samples to desired levels (e.g. 100 ng/ul), as much as possible.  Some samples may be highly concentrated.  Dilute these further with TE.