1. DNA Fingerprinting Lab by Bio-Rad

2. Equipment Micropipet—measures small volumes of liquid (  l –ml) 1000  l = 1 ml Microfuge tubes

3. Micropipets 1 ml = 1000  l Micropipets you will be using measure volumes between 2  l and 20  l –DO NOT TURN THE KNOB BEYOND THESE LIMITS!! 127127 = 12.7  l 025025 = 2.5  l

4. 172172 034034 023023 = 2.3  l 200200 = 20.0  l = 17.2  l = 3.4  l 1. 2. 3. 4.

5. Using a Micropipet To draw up fluid: –Turn the knob so that the window reads the correct volume –Gently tap the micropipet into a pipet tip in the tip box DO NOT PUT THE MICROPIPET DIRECTLY INTO FLUID WITHOUT A TIP!!! DO NOT TOUCH THE TIP WITH YOUR FINGERS OR ANYTHING ELSE!! –Push down the top yellow button until you feel the first “stop” –Place the tip of the pipet tip into the solution –Slowly release the button

6. Using a Micropipet To release fluid: –Place the tip of the pipet tip at the bottom of the microfuge tube –Push down the top yellow button past the first “stop” –Pull the tip out of the solution –Slowly release the button

7. Using a Micropipet To release pipet tip: –Hold micropipet over “waste” cup –Push the blue button on top of the pipet You must change tips in between each solution to prevent contamination!

8. Minicentrifuge—pools liquid, solids to bottom of centrifuge tube Samples of balanced rotor configurations

9. Agarose gel—porous Made of agarose and 1x TAE buffer Run in a gel box with 1x TAE buffer

10. Evolved by bacteria to protect against viral DNA infection Endonucleases = cleave within DNA strands 3,139 known enzymes Restriction Enzymes

11. Enzyme Site Recognition Each enzyme digests (cuts) DNA at a specific sequence = restriction site Enzymes recognize 4- or 6- base pair, palindromic sequences (eg GAATTC)

12. 5’ versus 3’ overhang Enzyme cuts  5’ GAATTC 3’ 3’ G 5’ 5’ G 3’ 3’ CTTAAG 5’ 5’ G 3’ 3’ CTTAA 5’ 5’ AATTC 3’ 3’ G 5’  Generates 5’ overhang

13. Common Restriction Enzymes EcoRI –Escherichia coli – 5’ overhang PstI –Providencia stuartii –3’ overhang 5’ GAATTC 3’ 3’ CTTAAG 5’ 5’ CTGCAG 3’ 3’ CACGTC 5’

14. The DNA Digestion Restriction Buffer provides optimal conditions – NaCl provides the correct ionic strength – Tris-HCl provides the proper pH (pH 8) – Mg 2+ is an enzyme co-factor

15. DNA Digestion Temperature Why incubate at 37  C? Body temperature is optimal for these and most other enzymes What happens if temperature is too hot or cool? Too hot = enzyme may be denatured Too cool= enzyme activity lowered, requiring longer digestion time

16. Analysis of Stained Gel Determine restriction fragment sizes – Create standard curve using DNA marker – Measure distance traveled by restriction fragments – Determine size of DNA fragments Identify the related samples

17. Molecular Weight Determination Size (bp) Distance (mm) 23,00011.0 9,40013.0 6,50015.0 4,40018.0 2,30023.0 2,00024.0 Fingerprinting Standard Curve: Semi-log