1. Restriction Analysis of Plasmids
Lab Details

2. 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

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. 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

5. 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

6. 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

7. 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)

8. HindIII
HindIII recognizes the following sequence:

9. 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

10. 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.

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. - +
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

16. 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!