1. WSSP-14 Chapter 3 Analyzing DNA –Restriction Digests © 2014 WSSP

2. p. 1-10 Cloning cDNA fragments into pTripleEX2 © 2014 WSSP

3. Restriction enzymes are used for cloning and analyzing DNA fragments © 2014 WSSP

4. 1 2 3 4 1234 ONs 1234 MPs 4.0 1.0 2.0 1.6 1.0 0.5 3.0 4.0 M1234 RI/HindIII 2.0 1.6 1.0 0.5 3.0 4.0 M1234 PstI

5. 1 2 3 4 1234 ONs 1234 MPs 3.0 4.0 2.0 1.0 2.0 1.6 1.0 0.5 3.0 4.0 M1234 RI/HindIII

6. Bacteriophage - "eaters of bacteria" © 2014 WSSP

7. Restriction enzymes - endonucleases, Cleave a specific DNA sequence Protect bacteria from phage infection, digest phage DNA after infection p. 3-1 GAATTC CTTAAG G CTTAA AATTC G © 2014 WSSP

8. Why don't the restriction enzymes degrade the cell DNA? © 2014 WSSP

9. Cellular DNA protected by methylases - block restriction enzyme activity p. 3-1 GAATTC CTTAAG G CTTAA AATTC G GAATTC CTTAAG M M Methylase GAATTC CTTAAG M M EcoRI © 2014 WSSP

10. DNA-binding proteins make non-covalent contacts to the DNA Methyl group CH 3 Methylation blocks binding by the protein

11. NOTE: DNA Methylation in bacteria has a different purpose then DNA methylation in eukaryotes Vertebrate CpG methylation – reduces gene expression in adult somatic cells, hypermethylation silences genes Genomic imprinting, X-chromosome inactivation, suppression of repetitive elements Bacterial Dam methylase (GATC) involved in mismatch repair, replication, and gene expression © 2014 WSSP

12. Each strain of bacteria has a specific set of restriction enzymes EcoRI from Escherichia coli BamHI from Bacillus amyloliqueraciens PvuI and PvuII are different enzymes from same strain. Originally purified by individual labs, Nathans, Smith Now supplied by companies - NEB, Promega p. 3-1 © 2014 WSSP

13. Sequence Recognition and cleavage: a) 5' overhangEcoRIGAATTCG OH p AATTC CTTAAGCTTAA p OH G b) 3' overhang KpnIGGTACCGGTAC OH p C CCATGGC p OH CATGG c) Blunt endSmaICCCGGGCCC OH p GGG GGGCCCGGG p OH CCC p. 3-2 © 2014 WSSP

14. DNA fragments with compatible sticky ends can be ligated together Example: NNG AATTCNNNNNGAATTCNNN NNCTTAA GNNNNNCTTAAGNNN Not NNGGGCCCNNNNNGGGCCCNNN NNCTTAA GNNNNNCTTAAGNNN Ligase +ATP OHP P P P © 2014 WSSP

15. DNA fragments with blunt ends can be ligated together Example: NNGAATTCNNNNNGAATTCNNN NNCTT AAGNNNNNCTTAAGNNN NNGAACCCNNNNNGAACCCNNN NNCTTGGGNNNNNCTTGGGNNN Ligase OHP P P P © 2014 WSSP

16. Sequence Recognition and cleavage: d) Degenerate: AvaII GGWCC:GGTCC, GGACC AvaI CPyCGPuG CTCGAG Py stands for pyrimidine- T or C CTCGGG Pu stands for purine - A or G CCCGAG CCCGGG CTCGAGCCCGGGCCCGAG GAGCTCGGGCCCGAGCCC DdeI CTNAG: CTAAG, CTGAG, CTCAG, CTTAG BbsI cleaves GAAGACNN CTTCTGNNNNNN p. 3-2 © 2014 WSSP

17. Recognition sites for the SfiI restriction enzyme p. 3-2 © 2014 WSSP

18. p. 3-3 © 2014 WSSP

19. Activity: in units which corresponds to a specified level of enzyme activity. NEB defines a unit as: “One unit of restriction endonuclease activity is defined as the amount of enzyme required to completely digest 1  g of substrate DNA in a total reaction volume of 0.05 ml in one hour using the NEB buffer provided.” p. 3-3 © 2014 WSSP

20. Before setting up a restriction digest check to make sure that you are using the proper conditions! Improper conditions can denature (inactivate) the enzyme or cause non-specific digestion (Star activity) Activity of an enzyme can change under different conditions: pH- 7.5, 8.0, 8.5 salt concentration- 20 mM, -150 mM divalent cations- Mg++ reducing reagent- DTT carrier protein-BSA temperature- 37C, RT, 60C Restriction enzymes are proteins with optimal conditions p. 3-4 © 2014 WSSP

21. p. 3-4 Information on Restriction Enzymes © 2014 WSSP

22. 4. Set up the Reaction: Add in the following order: Sterile ddH 2 O7.0  l 10 X restriction buffer 2.0  l Miniprep DNA (0.5  g) 10.0  l Enzyme (20 U/  l)1.0  l Total volume20.0  l The two most important rules in enzymes Always keep enzymes on ice or in a cooler. Always use a fresh tip when pipeting from the enzyme stocks. p. 3-5 © 2014 WSSP

23. Set up a master mix multiple digests: SingleMultiple Sterile ddH 2 O7.0  l35.0  l 10 X restriction buffer 2.0  l10.0  l Miniprep DNA (0.5  g) 10.0  l**none** Enzyme (20 U/  l)1.0  l5.0  l Total volume20.0  l 10.0  l Mix aliquot 10.0  l DNA p. 3-7 Never put anything into your yellow MP tube!!! © 2014 WSSP

24. 5. Incubate reactions at the appropriate temperature for the appropriate time. Usually 37˚C and incubate 1 hr or more. 6. If running on a gel: Add gel loading dye EDTA - Stops reaction. Dyes (BPB and XC) - to help see sample while loading and monitor electrophoresis Glycerol - so sample sits at bottom of the well p. 3-6 © 2014 WSSP

25. Using a restriction digest to map a plasmid p. 3-8 © 2014 WSSP

26. Map of the pTriplEX2 plasmid p. 3-9 cDNA Fragment © 2014 WSSP

27. Why not test cut the plasmid with SfiI? 2. Can’t tell if single cut or non insert 3. It is also expensive. p. 3-10 1. May have lost the site in cloning © 2014 WSSP

28. Theoretical PvuII-HF digests of plasmids p. 3-10 © 2014 WSSP

29. Why are there 3 bands in the uncut lane? p. 3-10 © 2014 WSSP

30. A real Digest gel of clones from the Duckweed cDNA library p. 3-11 © 2014 WSSP

31. 5. Does your digest look complete? Are the intensities of the bands proportional to the size? p. 3-12 © 2014 WSSP

32. Bands Generated from Partial Digests p. 3-19 © 2014 WSSP

33. 6. Is there a PvuII site in the insert? Are there more than two bands? p. 3-12 © 2014 WSSP

34. 6. Is there a PvuII site in the insert? Are there multiple bands? p. 3-13 © 2014 WSSP

35. 7. What is the size of the insert? p. 3-13 700 bp - 700 bp = 0 bp 2.9 Kb © 2014 WSSP

36. 8. Do the results from the digest and the PCR agree? p. 3-14 200 bp - 200 bp = 0 bp 700 bp - 700 bp = 0 bp © 2014 WSSP

37. 8. Do the results from the 20JM1.10 digest and the PCR agree? p. 3-14 600 bp - 200 bp = 400 bp 1100 bp - 700 bp = 400 bp © 2014 WSSP

38. 8. Do the results from the 20JM2.10 digest and the PCR agree? p. 3-14 1700 bp - 200 bp = 1500 bp 2200 bp - 700 bp = 1500 bp © 2014 WSSP

39. 8. How do we figure out the insert size from the 20JM4.10 digest? p. 3-14 1200 bp + 400 bp- 700 bp = 900 bp 2900 bp 1200 bp 400 bp © 2014 WSSP

40. 8. Do the results from the 20JM4.10 digest and the PCR agree? p. 3-14 1100 bp - 200 bp = 900 bp 1200 bp +400 bp- 700 bp = 900 bp

41. Enter the results of the RD gel and whether the clone should be sequenced into your school’s Google Docs Clone Report sheet © 2014 WSSP

42. Enter the conclusions into the Google Docs Clone report © 2014 WSSP