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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