Restriction Analysis of PlasmidsRestriction Analysis of Plasmids

Lab Details

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

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

• 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.

BamHI

+ BamHI

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)

One restriction site is arbitrarily determined to be the 0 point

The other restriction sites are given in reference to the 0 point

Plasmid B1250 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?

supercoiled

-

+

Nicked circle

Multimer

• 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!