Restriction Analysis of Plasmids Lab Details

Memory Jog What are plasmids? What are restriction enzymes? 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

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

Sample Problem #1 BamHI + BamHI 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

Sample Problem #2 Suppose you have another plasmid called plasmid B that has 2 restriction sites for BamHI: BamHI (0 bp) BamHI (700 bp) The other restriction sites are given in reference to the 0 point One restriction site is arbitrarily determined to be the 0 point Plasmid B 1250 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 BamHI (0 bp) BamHI (700 bp) 700 bp 550 bp

Lab Your task: Analyze 3 different plasmids (M, B, S) to determine The total size (in bp) of each plasmid How many restriction sites each plasmid has for the enzyme HindIII How far apart the restriction sites are from one another (if there is more than one)

HindIII HindIII recognizes the following sequence:

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

Uncut plasmids Uncut plasmids will also be run as a control (M0, B0, S0)– 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 allows us to know which bands in gel are due to uncut plasmids.

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

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

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

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.

- + 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? Multimer Nicked circle supercoiled

Pre Lab Quiz tomorrow! 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 Pre Lab Quiz tomorrow!