Restriction Analysis of Plasmids

1. Restriction Analysis of Plasmids Lab Details

2. Memory Jog • What are plasmids? • Small, circular pieces of DNA found in bacteria • What are restriction enzymes? • Enzymes that cut DNA at specific sequences • How can you create a recombinant plasmid? • Cut gene of interest and plasmid with the same restriction enzyme • Mix together and seal with ligase

3. Plasmid Mapping • In order to use a plasmid to create recombinant plasmids, we need to find out • Which restriction enzymes will cut a particular plasmid • How many places a restriction enzyme will cut a particular plasmid • How far apart restriction sites are

4. BamHI + BamHI Sample Problem #1 • Suppose you have a plasmid called plasmid A with the one restriction site for BamHI. • If you cut this plasmid with BamHI, how many fragments of DNA will you get? (think carefully!) • One– cutting the plasmid linearizes it. Plasmid A

5. BamHI (0 bp) The other restriction sites are given in reference to the 0 point One restriction site is arbitrarily determined to be the 0 point BamHI (700 bp) Sample Problem #2 • Suppose you have another plasmid called plasmid B that has 2 restriction sites for BamHI: Plasmid B 1250 bp

6. BamHI (0 bp) 700 bp BamHI (700 bp) 550 bp • How many fragments will result from digesting this plasmid with BamHI? • 2 different fragments • How big are the fragments? • 700 bp and 550 bp

7. Lab • Your task: Analyze 2 different plasmids (B, S or pGFP) to determine • The total size (in bp) of each plasmid • How many restriction sites each plasmid has for the enzyme HindIII or another restriction enzyme • How far apart the restriction sites are from one another (if there is more than one)

8. HindIII • HindIII recognizes the following sequence:

9. General Steps • Monday: Digest each of the DNA samples with enzyme for 30 minutes • Tues/Wed: Run the DNA samples on a gel • Thurs: Analyze gels

10. Uncut plasmids • Uncut plasmids will also be run as a control (labeled __“0”)– no HindIII added • An uncut plasmid is still circular  won’t move through the gel in the same way a linear molecule would • Running these controls helps us tell which bands in gel are due to uncut plasmids (so we don’t count them as fragments).

11. Uncut Plasmid Forms • Uncut plasmid are most likely to exist in one of 3 forms: • Supercoiled • Nicked • Multimer

12. Supercoiled Plasmid • When the plasmid gets twisted up • Moves faster through the gel than other forms of uncut plasmid because they are so compact

13. Nicked circles • Sometimes the plasmids get partly “nicked” (cut) by handling and refreezing • Causes it to unwind  can’t move through the gel as easily as the supercoiled form

14. Multimers • Sometimes plasmids get linked together when they are being copied in a bacteria • This multimer has twice as much DNA so it moves slower through the gel.

15. - Multimer supercoiled Nicked circle + Uncut Plasmids on a Gel • When you run the uncut plasmids on a gel, you will get a pattern of bands (at least 3-4) • Which band of DNA is • the supercoiled plasmids? • Nicked circles? • Multimers?

16. Now, walk through Monday’s Procedure • Homework = Set up Lab Notebook • Write a specific title (hint: plasmid and Enzyme names…) • Write an Introduction • Paste in Procedure (both pages) • make a flowchart of procedure