DNA-Fingerprint1 Procedure of DNA-Fingerprints

DNA-Fingerprint2 Tubes for each workgroup

DNA-Fingerprint3 Suspect 1 – 5 Tubes contain 10 µl of DNA sample 70 µl Enzyme mix 0,24 g Agarose 10 µl Marker 80 µl Loading buffer 10 µl CS DNA

DNA-Fingerprint4 1. Preparation of the Restriction

DNA-Fingerprint5 Attention! For each pipetting use a new pipet tip to avoid contaminations. Please snip all the tubes at least for 30 seconds for mixing.

DNA-Fingerprint6 1. Tubes with DNA-Samples DNA 10µl CS S 1 S 2 S 3 S 4S 5 10µl

DNA-Fingerprint7 1.1 Pipet 10 µl of enzyme mix into each tube DNA 10µl CS S 1 S 2 S 3 S 4S 5 10µl Enzyme- 10µl10µl 10µl 10µl 10µl 10µl mix (ENZ)

DNA-Fingerprint8 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.

DNA-Fingerprint9 3. Prepare Electrophoresis

DNA-Fingerprint10 Inserting of the sledge, the metal rods and the comb into the electrophoresis chamber 3.1 Preparing the electrophoresis chamber Metal rods Sledge Comb

DNA-Fingerprint 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.

DNA-Fingerprint 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 till it‘s solid.

DNA-Fingerprint 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.

DNA-Fingerprint14 4. Preparation of DNA-Marker Pipet 10 µl loading buffer (LB) into 10 µl DNA- marker (M).

DNA-Fingerprint15 5. Addition of 10 µl Loading Buffer Pipet 10 µl of the loading buffer into the CS and S1- S5. 10 µl

DNA-Fingerprint16 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

DNA-Fingerprint17 6. Filling the Wells Attention: Gel bags must not be pierced.

DNA-Fingerprint18 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.

DNA-Fingerprint19 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.

DNA-Fingerprint20 9. Analyse the gel CS S1 S2 S3 S4 S5 M Which of the suspects is the perpetrator?

DNA-Fingerprint21 9. Result