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

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

2. 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 Learning goals

3. 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. Key Ideas

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

5. Manipulatives help students visualize plasmid configurations supercoil Tips

6. 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. Tips

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

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

9. R- R+ 10 Kb Ladder 10 Kb Ladder 10 Kb Ladder Multimer Nicked Super Coiled 5 Kb Linear Fragment Linear Fragment

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

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

12. EvaGreenVsEtBr staining Ethidium Bromide staining of gels Stain = 10 minutes

13. Carefully slide the gel into the staining container • Cover gel with EtBrfor 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. Ethidium Bromide staining of gels

14. 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. What does Lab 4A “give” to us?

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