DNA extraction from tail clips

Overview

There are two methods of isolating DNA from the tail clip. The "dirty" method is to digest the tail in a Triton-based tail buffer. With this method, PCR is performed directly from the solution of digested tail. The "clean" method is to digest the tail in an SDS-based tail buffer, then extract the DNA using a phenol-chloroform DNA extraction protocol. This results in purified DNA that is then used for PCR. In general, we use the "dirty" method because it is quicker and cheaper and usually works.

Protocol

• Clip 5 mm of mouse tail using razor blade or scissors and place in a 1.5 ml microcentrifuge tube.
• Add 700 µl of Triton-based tail buffer (dirty method) or 700 µl of SDS-based tail buffer (clean method).
• Add 12.5 µl 20 mg/ml Proteinase K Incubate in 55°C shaker overnight at 250 rpms. Lay tubes horizontally to get maximum mixing.
• Store at -20°C or proceed to DNA purification
• If using the "dirty" method can use this directly for PCR
• If using the "clean" method, extract DNA using the phenol-chloroform purification protocol

Triton-based tail buffer 1 L

SDS-based tail buffer 1 L

Alternative: SDS/SSC tail lysis buffer

Phenol/Chloroform purification

• Add an equal volume (~700 µl) of equilibrated phenol (pH 7.9)
• Vortex 10 sec. Centrifuge 5 min. at 14,000 rpm in microfuge to separate layers
• Carefully transfer top aqueous layer to clean labeled tube
• Discard lower phenol layer in liquid waste jug
• Add equal volume (~600 µl) of 1:1 mix of phenol/chloroform:IAA (24:1) or just a 1:1 mix of phenol/chloroform
• Vortex 10 sec. Centrifuge 3 min 14,000 rpm to separate
• Transfer top aqueous layer to clean labeled tube
• Add equal volume (~500 µl) of chloroform
• Vortex 10 sec. Centrifuge 2 min. 14,000 rpm to separate.
• Transfer top layer to clean labeled tube
• Add 2 volumes (~800 µl) of rt 95% ethanol
• Gently invert the tubes until precipitate appears
• Can put in -20 freezer for 30 min to get better precipitation
• Centrifuge 15 min at 4 C at 14,000 rpm
• Pour off alcohol and let residual ethanol evaporate at rt or 37 C dry incubator
• Resuspend DNA in 50-100 microliters TE
• Incubate at 37 C overnight
• Normally get about 50 µg DNA/tail with an A260/280 ratio of 1.84

Alternative quick NaOH-based DNA purification method

• Cut 1-2 mm of tail (very small)
• Add 200-300 µl of 50 mM NaOH
• Heat to 95°C for 10 min and vortex
• Add 50 µl 1M Tris (pH8.0)
• Centrifuge 12,000 rpm for 6 min.
• Transfer supernatant to new tube DNA is ready for downstream analysis

Vacutainer SST Tube method

• Follow first three steps of protocol to collect tail and digest with proteinase K overnight
• Use BD Vacutainer SST Blood Collection Tubes (cat # 367986) 5.0 ml * 13 x100 mm
• Work in a biosafety cabinet
• Remove cap from tubes
• Pipette all of the tail digest (~700 µl) into vacutainer tube
• Add 500 µl phenol/chloroform/isoamyl alcohol (25:24:1) to tube
• Spin 3000 rpm (1690 rcf) at 4ºc for 10 min
• Add 500 µl 4ºc chloroform:isoamyl alcohol (24:1) to tube
• Spin 3000 rpm (1690 rcf) at 4ºc for 10 min
• Add an additional 500 µl 4ºc chloroform:isoamyl alcohol (24:1) to tube
• Spin 3000 rpm (1690 rcf) at 4ºc for 10 min
• Transfer aqueous top layer to a 1.5 ml microfuge tube. This will be about 500 µl. Try not to get the yellow stuff
• Add 500 µl 4ºc isopropanol to tube. Shake tube well, you should see DNA precipitate
• Spin 12,000 rpm (~13,000 rcf) at 4ºc for 5 min. Should see DNA pellet at bottom of tube
• Decant off isopropanol
• Add 500 µl 4ºc 70% ethanol. gently invert tube to wash the pellet
• Spin 12,000 rpm (~13,000 rcf) at 4ºc for 5 min. Should see DNA pellet at bottom of tube
• Decant off ethanol and air dry on bench for ~5-10 minutes
• Resuspend in ~200 µl TE buffer. Incubate at 37 ºc for a minimum of 2 hrs to overnight