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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 - PowerPoint PPT Presentation
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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
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