PRELAB DISCUSSION #12

Transcription

1 PRELAB DISCUSSION #12

2 ANNOUNCEMENTS KEY DATES: CH6C Labs: Dec2-8 th CH6 Write-up: pdf due 9 pm the evening before your CH6C Lab and hardcopy due at the beginning of CH6C lab. (The write up template outlining what needs to be turned-in is posted on blackboard content CH6 Materials CH6 Write-up Template. CH6 Write-up Addendum: pdf due 48 hrs after the end of your CH6C lab (each TF will arrange separately for the delivery of the hardcopy). It constitutes completing a three page addendum sheet based on your CH6C lab data. The sheet is posted on blackboard content CH6 Materials CH6 Addendum Sheet. You are encouraged to print that out and complete it DURING your lab time and turn it in (you do NOT need to turn in a pdf if you turn-in a hardcopy during lab time). But if you prefer, you can work on it until its due date. Lab Final: Dec 7 th and 8 th : Bring your calculator. Will focus on assessing your understanding of the experimental techniques covered in 421 labs throughout the semester in terms of collecting, analyzing, and interpreting data

3 PART I REFLECTING ON CH6AB WK 1 What did you do? (procedure) Isolated two plasmids (A&B) from Ecoli containing the rel gene inserted in two opposite orientations with respect to ORF Performed UV Absorbance measurements at 260 and 280 nms on the isolated plasmids. Performed agarose gel electrophoresis on the isolated plasmids. Why did you do it? (purpose) To obtain DNA plasmid containing the desired gene (CH6C,422) To perform restriction digest to create a restriction map of the plasmids To determine the concentration of the purified DNA plasmids

4 PART II PREPARING FOR CH6C WK 2 What will you do? (procedure) Perform restriction digests using Ahd I and Pvu II endonucleases Perform agarose gel electrophoresis on digested plasmids Why will you do it? (purpose) To create a restriction map for the isolated plasmids To determine the identity of each plasmid (which one is pgem3-rel (gene in reverse orientation to ORF) and which one is pgem4- Rel (gene in correct orientation to ORF)

5 YOUR PLASMIDS (A&B) Amp R SP6 Promoter pgem3-rel 5.27 Kb Rel Amp R SP6 Promoter pgem4-rel 5.27 Kb Pvu II 0.55 Rel Ahd I 3.57 ori Pvu II 2.50 Pvu II 1.92 Ahd I 3.57 ori Pvu II 2.50 Each plasmid contains: Rel gene Ampicilin resistence gene SP6 Promoter Origin of replication Restriction enzyme recognition sites

6 RESTRICTION ENDONUCLEASES (RE) Enzymes that recognize and digest DNA by cleaving phosphodiester bonds between nucleotides Genomic DNA is protected from digestion in the cell by DNA methylation There are three types of endonucleases: 1. Type I: Cleave DNA ast sites >1000 bps from the recognition site (require ATP) 2. Type II: Cleave DNA within a specific short sequence of bases called palindromes (does NOT require ATP) 3. Type III: Cleave DNA ~25 bp from recognition sequence (require ATP)

7 Restriction Endonucleases (RE) used in CH6C Ahd I: restriction site Puv II: restriction site Ahd I cuts the double strands asymmetrically, leaving protruding ends. These protruding bases are referred to as sticky ends. PuvII enzyme cuts the double strands symmetrically, leaving blunt ends The recognized sequences are the same on both strands when each strand is read 5 -> 3. Such symmetrical sequences are called palindromes.

8 PRODUCTS OF A RESTRICTION DIGEST Starting with a circular DNA (like your plasmids): If there is a single recognition site for a RE and the DNA is incubated with the RE: DNA cut once linear DNA size same as circular DNA If there are multiple recognition sites for a RE and the DNA is incubated with the RE: # cuts= # restriction sites= # of fragments produced (total size of fragments = size of circular DNA) Multiple REs can be used to digest a single plasmid simultaneously as long as the reaction conditions for the digestion are compatible.

9 DETERMINING THE NUMBER and SIZE of DIGESTED FRAGMENTS Run an agarose gel (typically 0.5-2%) Percentage determined by the expected fragment sizes higher percentage for smaller fragments Visualize DNA bands What s going to determine the location of the DNA bands on the gel? Size (# of bps): small pieces migrate faster, farther than bigger pieces. Conformation (shape): Comparing 3 pieces of DNA that are the same size. Supercoil < Linear < Nicked (relaxed) circular Charge: DNA is negative because of phosphate groups and have uniform m/e

10 DETERMINING THE NUMBER and SIZE of Plot migration distance in mm (relative migration-r m ) vs log Size (bp) of standards Determine the best linear fit to the data and use this line (or the graph) to find the size of the fragments Find the total size of the plasmids by adding up fragment sizes and confirm that it has the expected circular plasmid size. DIGESTED FRAGMENTS

11 CREATING RESTRICTION MAPS A restriction map is a physical map of a piece of DNA showing recognition sites of specific restriction enzymes separated by lengths marked in numbers of bases The pattern of DNA bands is characteristic for a specific DNA sample and the restriction enzymes used to cleave it. A banding pattern can be referred to as a DNA fingerprint. because it is unique to that particular DNA (and the combination of restriction fragments).

12 CH6C Workflow PRELAB: Calculate the amount in μl) of DNA you need for each digest based on the DNA concentration you have determined in CH6B (You need ~ 0.5 μg) Double check volume calculations from your DNA stock for each restriction digest set-up (total reaction volume 10 μl) Record this info in your worksheet and your notebook Prepare restriction digest reactions Record these set-up tables in your worksheet Cast a 1% agarose gel while the digest is going using a 10 well comb Prepare gel samples (Total 12 μl 10 μl digest + 2 μl 6X sample buffer) and electrophoresis chamber Load samples ( 6 digests and 1 Linear DNA Minnesota Markers) and run gel (be careful about the running direction- DNA is (-)) Stain in EtBr for 15 min, destain in water 2 min, and image gel on UV gel doc (take a picture and include it in your worksheet. SAVE PLASMID for BI/CH422 CH8

13 CH6 Addendum Sheet

14 CH6 Addendum Sheet

15 CH6 Addendum Sheet

16 Example of a Restriction Digest Plasmid px (6000 bps) digested with REs A, B, and C Based on the restriction map shown on the right complete the table below Reference point (start and end point of digest) Digestion with Enzyme A Enzyme B Enzyme C Enzyme A+B Enzyme A+C Enzyme B+C Enzyme A+B+C # of fragments Fragment Sizes (bps)

17 Enzyme A

18 Enzyme A

19 Enzyme B

20 Enzyme B

21 Enzyme C

22 Enzyme A + B

23 Enzyme A + B

24 Enzyme A + C

25 Enzyme B + C

26 Enzyme A + B + C

27 Enzyme A + B + C

28 Example of a Restriction Digest Plasmid px (6000 bps) digested with REs A, B, and C Based on the restriction map shown on the right complete the table below Reference point (start and end point of digest) Digestion with # of fragments Fragment Sizes (bps) Enzyme A 3 1,400, 1,500, 3,100 Enzyme B 2 2,000, 4,000 Enzyme C 1 6,000 Enzyme A+B 5 (4 bands) 1,400, 500, 1,000(2), 2,100 Enzyme A+C 4 1,400, 1,500, 2,100, 1,000 Enzyme B+C 3 2,000, 1,100, 2,900 Enzyme A+B+C 6 (4 bands) 1,400, 500, 1,000(3), 1,100

29 Example of a Restriction Digest Plasmid px (6000 bps) digested with REs A, B, and C Digestion with # of fragments Fragment Sizes (bps) Enzyme A 3 1,400, 1,500, 3,100 Enzyme B 2 2,000, 4,000 Enzyme C 1 6,000 Enzyme A+B 5 (4 bands) 1,400, 500, 1,000(2), 2,100 A B C A + B A + C B + C A + B + C Undi gest ed DNA lad der Enzyme A+C 4 1,400, 1,500, 2,100, 1,000 Enzyme B+C 3 2,000, 1,100, 2,900 Enzyme A+B+C 6 (4 bands) 1,400, 500, 1,000(3), 1,100

30 RFLP and DNA Fingerprinting RFLP (Restriction Fragment Length Polymorphism) analysis RFLP+PCR for DNA Fingerprinting Applications Disease detection/prevalence Forensics Paternity

31 Sickle-Cell RFLP 31

32 DNA fingerprinting in a murder case PCR amplify small amounts of DNA from crime scene Digest DNA and compare pattern of bands DNA fingerprint

33 DETERMINING PATERNITY Which one is the father? F1 or F2?