Restriction Analysis and Digestion of Lambda DNA

1. Restriction Analysis and Digestion of Lambda DNA

2. DNA is tightly packaged into chromosomes which reside in the nucleus

3. Model of DNADNA is Comprised of Four Base Pairs

5. DNA Restriction Enzymes • Used by bacteria to protect against viral DNA infection • Endonucleases = cleave within DNA strands • Over 3,000 known enzymes

6. Bacteriophage Lambda

8. Restriction Digestion and Analysis of Lambda DNA

9. Enzyme Site Recognition Restriction site Palindrome • Each enzyme digests (cuts) DNA at a specific sequence = restriction site • Enzymes recognize 4- or 6- base pair, palindromic sequences (eg GAATTC) Fragment 2 Fragment 1

10. Common Restriction Enzymes EcoRI – Eschericha coli – 5 prime overhang Pstl – Providencia stuartii – 3 prime overhang

11. Your tasks: • Cut lambda DNA into a series of fragments using restriction enzymes • To separate and sort a large group of DNA molecules according to theirsize

12. Important note: First add DNA, then restriction buffer, and then the enzymes to the tubes. Use a fresh pipette tip for restriction buffer and each enzyme.

13. The DNA Digestion Reaction Restriction Buffer provides optimal conditions • NaCI provides the correct ionic strength • Tris-HCI provides the proper pH • Mg2+ is an enzyme co-factor

14. Place the sample tubes in a 37°C water bath or oven for approximately 30 minutes • While you are waiting, this a good time to cast your agarose gel

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

16. Part 1. Prepare Your Samples for Electrophoresis • Add 2.0 μl of sample loading dye to each of the tubes marked L, P, E, and H in the foam tube holder. Use a fresh tip with each sample to avoid contamination

17. Part 2. Set Up Your Gel Electrophoresis Chamber • . Pour enough buffer into the box until it just covers the wells of the gel by 1–2 mm

18. Part 3. Load your Samples and Run them by Electrophoresis • Pipette 10 μl from each tube (M, L, P, E, and H) into separate wells in the gel chamber • Important • Electrophorese at 100 V for 30–40 minutes

19. Restriction Fragment Length Polymorphism RFLP PstI EcoRI GAATTC GTTAAC CTGCAG GAGCTC Allele 1 1 2 3 CGGCAG GCGCTC GAATTC GTTAAC Allele 2 3 Fragment 1+2 Different Base Pairs No restriction site M A-1 A-2 Electrophoresis of restriction fragments M: Marker A-1: Allele 1 Fragments A-2: Allele 2 Fragments