Ch. 4A: Making Sure You’ve Got What You Need

Ch. 4A: Making Sure You’ve Got What You Need

Learning goals

Describe why it is important to verify products created in the genetic engineering process

Predict the relative speed of DNA restriction fragments and plasmids through a gel during gel electrophoresis

Separate and identify DNA restriction fragments and

plasmids using gel electrophoresis

Key IdeasThe multistep process that is used to clone a gene

results in multiple products◦Need to verify that you have the recombinant plasmid you

need. DNA fragments and plasmids can be separated by gel

electrophoresis. Loading dye helps monitor the progress of the gel

electrophoresis procedure. DNA ladder helps determine the sizes of unknown

pieces of DNAGel is stained in order to show the location of the

DNA fragments and plasmids.

Tips

Discuss the selective properties of the agarose matrix varying with different agarose concentrations, as well as how fragment size may determine concentration.

Tips

Manipulatives help students visualize plasmid configurations

supercoil

Tips

DNA ladder looks like loading dye, students sometimes mistake it for loading dye.

Loading dye is the same as Solution 2.

Gels from Lab 1 can be reused if the dyes are allowed to “run off” the end of the gel, or gels are placed in buffer overnight in the fridge.

If you run short on time, gels can be placed in a ziplock baggie then in the fridge with a little 1xSB. Later in the day you (or student) can put it back in a tray and finish running the gel.

Restriction analysis of pARA-R

Restriction fragments after digest with Hind III and BamH I

Biotech Experience

806 bp

BamH IHind III

BamH I Hind III

4,496 bp

Different Structural Forms

circle

“nicked-circle”

“multimer”

Different structural forms produce different bands.

Nicked Circle

Supercoiled

Linear

From Michael Resenz ppt

10 K

b La

dder

5 Kb

MultimerNickedSuper Coiled

Linear Fragment

R- R+

Linear Fragment

10 K

b La

dder

10 K

b La

dder

Restriction analysis of pARA-R

Prediction for restriction gel

M R+ R-

500

1000

1500

20003000400050008000

10000

M R+ R-

Biotech Experience

Go to pg 63 and complete Lab 4A - Verification of the Recombinant Plasmid Using Gel Electrophoresis

Ethidium Bromide staining of gels

EvaGreen Vs EtBr stainingStain = 10 minutes

Ethidium Bromide staining of gels Carefully slide the gel into the staining container Cover gel with EtBr for 10 min. gently swirling

periodically. Pour the EtBr back into the aluminum covered

bottle. Destain the gel by covering with distilled water for

10 min. gently swirling. Carefully pour the water out of the tray - sink ok Carefully place the gel on the glass plate of the

transilluminator. Use a highlighter to label the bag.

What does Lab 4A “give” to us?Did the restriction enzymes digest the

pARA-R plasmid as expected?Allows visualization of digest products

– are they what we expected?Allows visualization of unique banding

resulting from non-digested plasmids.

M R- R+ M R- R+

Left end of the leading edge ofthe well may have been damaged,resulting in the uneven edges ofthe bands

Photo taken with digital camera Photo taken with doc. apparatus