Confirmation of pARA–R Restriction Digest

Laboratory 4a

Overview

Purpose:Examine the restriction fragments that result from the double digestion of pARA – R

By BamH I and Hind IIIPerform gel electrophoresis to visualize DNA plasmids

Introduction

DNA fragments move at different speedsSmaller = faster

Larger = slower

Take digested and undigested plasmid samples

Undigested = control (A-)

2-3 bands may appear

Plasmids isolated form cells may exist in several forms:

Introduction

Nicked-circleBreak in sugar-phosphate backbone

Moves slower than supercoiled

Multimer Replicated plasmids that remain linked together

Move the slowest

SupercoiledCompact molecule

Move quickly through gel

Different Structural Forms

circle

“nicked-circle”

“multimer”

Different structural forms produce different bands.

Nicked Circle

Supercoiled

Linear

Materials

ReagentsPlasmid samples

A-

A+

0.8% agarose gel

5 x loading dye

1 x SB

DNA size marker (25 ng/μL)

Equipment & SuppliesP-20 micropipette and tips

1.5 mL microfuge tubes

Electrophoresis equipment

Power supply

Plastic microfuge tube rack

Markers

Trans-illuminator

Camera set-up

Tube floats

Staining trays

What will you need to do?

Agarose gel

Aliquot:10 μL of 1 Kb ladder (marker) for each group

Methods

Methods3 tubes

A-

A+

DNA marker

Add 4 μL loading dye to tubes A- and A+Loading dye increases density so DNA will sink

Pump pipette to mix samples

New tip for each plasmid

Prepare the gel

Load samples

Methods

Loading samples:Use clean tip and pipette 20 μL of “DNA size marker”

Dispense as directed in Lab 1

Using clean tip load 24 μL A- into adjacent well

Using clean tip load 24 μL A+ into adjacent well

Connect leads and turn onAs directed in Lab 1

Let run about 30-40 minutes

Conclusions

• pARA–R – Should see 2 – 3 DNA bands

• 3 bands in A-

• 2 bands in A+– Has enzymes

Restriction analysis of pARA-R

Prediction for restriction gel

M A+ A-

500

1000

1500

20003000400050008000

10000

MA+A-

Bruce Wallace