Ch. 4A: Making Sure You’ve Got What You Need. Learning goals Describe why it is important to...
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Transcript of Ch. 4A: Making Sure You’ve Got What You Need. Learning goals Describe why it is important to...
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 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.
Tips
Discuss the selective properties of the agarose matrix varying with different agarose concentrations, as well as how fragment size may determine concentration.
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
Lad
der
5 Kb
MultimerNickedSuper Coiled
Linear Fragment
R- R+
Linear Fragment
10 K
b
Lad
der
10 K
b
Lad
der
Restriction analysis of pARA-R
Prediction for restriction gel
M R+ R-
500
1000
1500
20003000400050008000
10000
M R+ R-
Biotech Experience
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.