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

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Presentation Transcript
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

Learning goals
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.

Key Ideas
slide4

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

Tips
slide6

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
slide7

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

different structural forms

circle

“multimer”

Nicked Circle

Different Structural Forms

Linear

Supercoiled

“nicked-circle”

Different structural forms produce different bands.

From Michael Resenzppt

slide9

R-

R+

10 Kb Ladder

10 Kb Ladder

10 Kb Ladder

Multimer

Nicked

Super Coiled

5 Kb

Linear Fragment

Linear Fragment

slide10

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

slide11

Go to pg 63 and complete

Lab 4A - Verification of the Recombinant Plasmid Using Gel Electrophoresis

ethidium bromide staining of gels1

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
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.

What does Lab 4A “give” to us?
slide15

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