FINGERPRINTING?PROTOCOL?(AGAROSE?GEL)

FINGERPRINTING PROTOCOL (AGAROSE GEL) *

Restriction digests consist of:

15.75 ml ddH₂ O

1 µl 10 X buffer B (Boehringer Mannheim)

0.25 µl HindIII (40 U/µl)

3 µl DNA

Set up digestions in 96 well plates. Incubate at 37℃ for 4.5 hours. This can be done in a thermocycler.

After digestion, a brief centrifugation will collect DNA at the bottom of wells. Seal plates with foil tape and store at 4℃ if necessary.

Prepare 1% agarose gels in 1X TAE: Cool molten agarose to 46℃ in a water bath with occasional stirring. Pour into 20X25 cm UV transparent trays. Insert comb. Use 200 ml of molten agarose. After gel is solidified, remove comb and place in electrophoresis unit with enough 1X TAE buffer to cover the gel. Load 1.75 µl of the restriction enzyme digestion/ loading dye mixture into gel, with 1 µl of a standard marker in the first and every fifth well.

Marker is a mixture of 1 kb ladder (Life Technologies) and both Marker II and Marker III (Boehringer-Mannheim) in these proportions: 0.83 µl (1=B5g/µl), 3.33 µl (250 ng/µl) Marker II, 3.33 µl (250 ng/µl) Mar= ker III, 92.51 µl TE, 25 µl loading dye. Immediately before electrophoresis, remove 20µl of this mixture to a separate tube, dilute by adding 17 µl of TE and 3 µl of loading dye, and incubate at 60℃ for 5 minutes.

Electrophorese samples at 56V for 14 hours.

Stain with 500 ml of a 1: 10,000 dilution of Vistra Green in 1X TAE and agitate in the dark for 10-15 minutes. Two gels can be stained at one time and up to four gels can be stained using the same stock of dye. Store diluted stains in a foil covered container at 4℃. After staining, image gels using Fluorimager with the following scan settings: pixel size: 200 µm; digital resolution: 16 bits; detection sensitivity: high; PMT voltage: 950 V; filter :530 nm. Crop gel images and then convert them from the proprietary 16 bit Molecular Dynamics format to 8 bit TIFF images. Transfer by ftp to Unix workstations for band calling and contig building.