DNA fingerprintingYicheng Cao,



BAC DNA preparation

Miniprep with AutoGen 740

Innoculate 3-4 ml LB with 20µg/ml chloramphenicol

Grow at 37 °C overnight with shaking

Isolate BAC DNA with AutoGen 740

Resuspend BAC DNA in 50 µl TE pH8.0 or 50 µl ddH2O Miniprep with Qiagen kit

Check the quality and quantity of miniprep BAC DNA

Digest 5 µl BAC DNA with Hind III

Run in 1% agarose gel

Stain gel with ethidium bromide

Photograph the gel with Stratagene Eagle Eye II Still video system

If DNA was not good enough, repeat the procedure.

Fingerprinting Gel

Equipment and materials:

Stratagene sequencer - CastAwayTM gel system with gasket lubricant(Cat # 401075)

Stratagene castAwaytM precast gels (Cat # 401092): 4.5% precast polyacrylamide, 1xTBE, 7 M urea, with 32 preformed wells

High voltage power supplier (> 2000 V)

Stratagene castAwaytM gel dryer

Molecular dynamics storage phosphor screen (36 X 43 cm) Marker preparation

Digest lambda DNA with Hinf I (or use precut DNA)

Label: 5.0 µl lambda/Hinf I (10ng/ul)

4.0 µl 5X AMV-RT buffer (USB, Cat# 70218)

0.5 µl 32p- dATP

2.5 µl 3dNTP (-dATP) (0.33 mM each)

0.5 µl AMV-Reverse transcriptase (USB, #700412)

7.5 µl ddH2O

Total 20 µl.

incubate at 42 °C for 15-20 min. Add 20 µl ddH2O and 80 µl formamide mix (USB sequencing reaction stop solution, Cat.#70704). Aliquot and store at -20 °C. Boil 5 min and transfer to ice before use. Load 1 µl per lane. Sample preparation

Ban I - Msp I double digestion Make mixture: 2.0 µl New England BioLab buffer 2 0.5 µl Ban I (NEB, Cat# 118) 0.5 µl Msp I (NEB, Cat# 116) 0.25 µl RNase A 6.75 µl ddH2O (for AutoGen miniprep) or 13.75 µl ddH2O (for Qiagen miniprep) 10 µl BAC DNA (AutoGen miniprep) or 3 µl BAC DNA (Qiagen miniprep) Incubate at 37 °C for 1 hr. Add 50 µl 100% ethanol, vortex, incubate at room temperature for 3 min, and spin at max speed in a desktop centrifuge for 5 min. Air dry the pellets.

Label Make mixture:

10.0 µl 5X AMV-RT buffer

1.0 µl 32p-dATP

1.0 µl 3dNTP (-dATP) 3.3 mM each

0.5 µl AMV-reverse transcriptase

38.0 µl ddH2O

Total 50 µl for 1 gel (21 samples). Add 2 µl labelling mixture to each sample, mix very well and keep at 42 °C for 15 -20 min. Add 4 µl of formamide mix (Stratagene sequencing reaction stop solution). Boil 3-5 min aand keep on ice immediately. Load 1 µl(Qiagen prep) - 2 µl (AutoGen prep) High resolution denaturing gel (Stratagene precast sequencing gel (cat.# 401092)

Loading pattern: N M S C S M S S S M S S S M S S S M S S S M S S S M S S S M N N

M = Marker S = Sample to be analyzed on the gel C = Internal Control (loaded in a different lane on a different gel) N = any DNA sample (added to prevent lane bending)

Gel running Set up gel box at room temperature Running buffer: 1 X TBE Prerunning time: 25 min Run at consistent Watts, 30 Watts per gel Running time: about 85 min; run until the first dye (Bromophenol blue) arrives at low buffer reservoir. (The first dye run ~40 cm.) Run 4-6 gels (80-120 samples don't including internal controls) per day.

Fix gel in 10% acetic acid - 10% methanol for 15-20 min. Rinse gel with dH2O for couple of min to remove urea.

Put gel into Stratagene gel dryer to automatically dry the gel (about 40 min).

Cover the gel with handi-wrap plastic film , mark the gel number and date carefully and set it into Molecular Dynamics storage phosphor screen cassette and exposure for ~ 40 hrs.

Gel imaging

Scan the gel image with Molecular Dynamics PhosphorImager

Pre-process the gel image with ImageQuant (IQ) software

Cut the raw image into smaller size and save

Convert the above new image from 16-bit to 8-bit tiff format

Edit fingerprint image with Image 2.1 or 3.x

For Image 2.1, the image needs to be modified first with XV (John Bradley)

Invert the image by 90° so that sample bands run from left to right

Set the image size from "a x b" to "2a x (b/2)" or "a x (b/4)"

Use color edit function of XV to make a "reverse vedio" of the image (required by image 2.1) A processed sample image is available for .

If using image 3.x, the 8-bit tiff file from ImageQuant can be used directly. Refer to the for more information and a step by step tutorial.

Analyze fingerprint image with FPC 2.5