DNA fingerprinting
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DNA fingerprinting
Yicheng 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 ddH2 O
- Miniprep with Qiagen kit
- Miniprep with AutoGen 740
- 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 ddH2 O Total 20 µl.
incubate at 42 °C for 15-20 min. Add 20 µl ddH2 O 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 ddH2 O (for AutoGen miniprep) or 13.75 µl ddH2 O (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 ddH2 O
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 dH2 O 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.
- Loading pattern:
- Equipment and materials:
- 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 downloading .
- If using image 3.x, the 8-bit tiff file from ImageQuant can be used directly. Refer to the Sanger Center for more information and a step by step tutorial.
- For Image 2.1, the image needs to be modified first with XV (John Bradley)
- Analyze fingerprint image with FPC 2.5