1. Ch. 4A: Making Sure You’ve Got What You Need
Student Guide pg 55-68; Teacher Guide pg

2. 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

3. Key Ideas
The 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 DNA
Gel is stained in order to show the location of the DNA fragments and plasmids.

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

5. Manipulatives help students visualize plasmid configurations
Tips
Manipulatives help students visualize plasmid configurations
supercoil

6. 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.

7. Restriction fragments after digest with Hind III and BamH I
Restriction analysis of pARA-R
Biotech Experience
Restriction fragments after digest with Hind III and BamH I
BamH I
Hind III
4,496 bp
806 bp
BamH I
Hind III

8. Different Structural Forms
circle
“multimer”
Nicked Circle
Linear
Supercoiled
“nicked-circle”
Different structural forms produce different bands.
From Michael Resenz ppt

9. R- R+ 10 Kb Ladder 10 Kb Ladder 10 Kb Ladder Multimer Nicked
Super Coiled
5 Kb
Linear Fragment
This image was taken with a digital camera, zooming, low light settings, and super macro settings. This is NOT an image from the gel imager that was supplied with the kit. This is what the gel will look like when fluoresced under UV light.
Linear Fragment

10. Prediction for restriction gel
Restriction analysis of pARA-R
Biotech Experience
Prediction for restriction gel M R+ R- M R+ R-
500
1000
1500
2000
3000
4000
5000
8000
10000

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

12. Ethidium Bromide staining of gels
EvaGreen Vs EtBr staining
Stain = 10 minutes

13. 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.

14. 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.

15. Photo taken with digital camera Photo taken with doc. apparatus
Photo 2 added
Left end of the leading edge of
the well may have been damaged,
resulting in the uneven edges of
the bands