Unformatted text preview: Bi 1x, Spring 2010
o Agarose gel electrophoresis
• Your digestion reactions from last week
6X DNA loading dye
1 kb and 100 bp ladders (pre-stained)
HindIII digested lambda DNA (pre-stained)
pZE21-lacZ DNA (pre-stained)
Your PCR reactions Background
Agarose gel electrophoresis is a powerful separation method frequently used to analyze DNA
fragments generated by restriction enzymes. The agarose gel consists of microscopic pores that act
as a molecular sieve. Samples of DNA are loaded into wells made in the gel during casting, as
shown in the figure below. http://ocw.mit.edu/OcwWeb/Biological-Engineering/20-109Fall-2007/Labs/detail/mod1_2.htm Since DNA has a strong negative charge at neutral pH, it migrates through the gel towards the
positive electrode during electrophoresis. The DNA molecules are separated in the gel according to
their size and shape. Linear DNA molecules are separated according to their size. The smaller the
linear fragment, the faster it migrates. If the size of two fragments are similar or identical, they will
migrate together in the gel. If DNA is cleaved many times, the wide range of fragments produced
will appear as a smear after electrophoresis.
Circular DNAs such as plasmids are supercoiled. Supercoiled DNA has a more compact and
entangled shape (like a twisted rubber band) than its corresponding non-supercoiled forms (linear,
nicked and relaxed circles). When supercoiled DNA is cleaved by a restriction enzyme just once it
unravels to its linear form. If supercoiled DNA is nicked (a phosphate bond is broken anywhere in
the molecule, in either strand) it completely unravels to form a circle. Under the electrophoresis
conditions being used in this experiment, supercoiled DNA migrates faster than its linear form and
linear DNA migrates faster than its nicked circular form. (EDVOTEK EDVO-kit #102 Manual) Page 1 of 2 In order to determine the size of your DNA from the migration distance, you will use “DNA
ladders,” which are molecular weight standards that consist of DNA fragments of known sizes. The
ladders you will use today are shown below:
DNA Ladders a) Example of 1 kb DNA ladder on a 0.8% agarose gel, stained with EtBr. b) Example of the 100bp DNA ladder on a 1.3% agarose gel, stained with EtBr. Protocol:
You will run the following samples on a 1% TAE-Agarose gel (prepared by your TAs):
a. 100 bp DNA ladder
b. EcoRI/HindIII lambda DNA double digest (from session 1)
c. HindIII digest of lambda DNA (as a reference)
d. pZE21-LacZ – HindIII single digest (from session 1)
e. pZE21-LacZ – KpnI/HindIII double digest (from session 1)
f. Undigested pZE21-LacZ (as a reference)
g. PCR product
h. PCR no template control
i. PCR no primer control
j. 1 kb DNA ladder
1. Mix 10 µl of each of your restriction digests with 2 µl of 6X DNA loading dye. Mix by
pipetting up and down.
a. DNA loading dye consists of glycerol—which will help your solutions sink into the
wells of the agarose gel—and reference dyes that help you determine how far your
samples have migrated.
2. Mix 10 µ l of each of your PCR reactions with 2 µ l of 6x loading dye. Mix by pipetting
up and down.
3. Load your samples on the gel in the order listed above. Be sure to write it in your
a. For samples that are supplied to you pre-stained (ladders, references), load 5 µl.
b. For your own restriction digests from session 1, load 10 µl of sample/dye mixture.
c. To load the gel, place your pipette beneath the buffer surface layer, part-way into
your target well. Very slowly depress the pipette plunger to load your sample. As
you depress the plunger, you will notice the sample fall into the well. Do not rush!
This may cause the sample to flow out of the well.
d. Loading samples onto agarose gels is tricky! Ask your TA to demonstrate how to
load a gel before attempting to do it yourself.
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This document was uploaded on 01/03/2012.
- Fall '09