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Procedure of DNA-Fingerprints
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Tubes for each workgroup
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Suspect 1 – 5Tubes contain 10 µl of DNA sample
70 µl Enzyme mix
0,24 gAgarose
10 µl Marker
80 µl Loadingbuffer
10 µl CS DNA
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1. Preparation of the Restriction
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Attention!
• For each pipetting use a new pipet tip to avoid contaminations.
• Please snip all the tubes at least for 30 seconds for mixing.
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1. Tubes with DNA-Samples
DNA 10µl 10µl 10µl 10µl 10µl
CS S 1 S 2 S 3 S 4 S 5
10µl
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1.1 Pipet 10 µl of enzyme mix into each tube
DNA 10µl 10µl 10µl 10µl 10µl
CS S 1 S 2 S 3 S 4 S 5
10µl
Enzyme- 10µl 10µl 10µl 10µl 10µl 10µl mix (ENZ)
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2. Restriction Digestion
Place the tubes in the floating rack and incubate for 20 min at 37 °C in a water bath or an heating incubator.
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3. Prepare Electrophoresis
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Inserting of the sledge, the metal rods and the comb into the electrophoresis chamber
3.1 Preparing the electrophoresis chamber
Metal rods
Sledge
Comb
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3.2 Produce the agarose gel (0,8%)
• Give 0,24 g agarose + 30 ml TAE-buffer into a 250 ml Erlenmeyer flask.
• Boil the Erlenmeyer flask shortly in the microwave, swirl it and boil it shortly again.
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3.3 Pouring of the gel
Pour the gel at 55 °C into the gel box.
Attention:
•Test the temperature on the back of your hand.
•Don‘t move the gel tillit‘s solid.
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3.4 Preparing of the chamber
• After the gel has become solid, remove the metal rods of the electropho-resis chamber.
• Fill the chamber with 270 ml of TAE-buffer to cover the gel.
• Pull the comb.
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4. Preparation of DNA-Marker
Pipet 10 µl loading buffer (LB) into 10 µl DNA-marker (M).
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5. Addition of 10 µl Loading Buffer
Pipet 10 µl of the loading buffer into the CS and S1- S5.
10 µl10 µl
10 µl
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Pipet 20 µl of each sample and 20 µl DNA marker (M) into to the wells.
6. Filling of the Wells
Remember the sequence!
Lanes: fill from left to right
Left lane stays free
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6. Filling the Wells
Attention:Gel bags must notbe pierced.
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7. Gel-Electrophoresis (at 120 V ca. 30 min)
Stop the electrophoresis when the blue band has migrated to 1 cm before the end of the gel.
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8. Colouring of the DNA
• Transfer the gel into the colouring bowl and cover it with 60 ml of staining solution for 2 min.
• Put the staining solution back into the Erlenmeyer flask.
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9. Analyse the gel
CS S1 S2 S3 S4 S5 M
Which of the suspects is the perpetrator?
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9. Result