Download protocol: restriction endonuclease digestion/analysis of

BIOL 3716: Molecular Microbiology I
PROTOCOL: RESTRICTION ENDONUCLEASE
DIGESTION/ANALYSIS OF PCR PRODUCTS
(v. 091508)
Purpose: To analyze the restriction endonuclease digestion patterns of purified PCR products
Source/Background Information:
A single PCR-generated band in a gel does not always indicate a single amplification product.
One way to quickly determine if there is more than one product is to digest the PCR product with
restriction endonucleases. For example, following restriction endonuclease digestion, if DNA
fragments are visualized by gel electrophoresis in addition to the original PCR product, then the
amplification process likely produced at least two distinct DNA products. Furthermore, if
multiple restriction endonucleases are employed, a map of “cut” sites can be generated for the
PCR products.
Various brands of restriction endonucleases can be used for this procedure. For the present
protocol, specially formulated restriction endonucleases (FastDigest) will be employed that allow
complete digestions of PCR products to occur following 20 minutes of incubation. For
background information on the use of FastDigest restriction endonucleases, see the following
URL: http://www.fermentas.com/fastdigest/index.html.
Materials Needed:
Per Student Group
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PCR mixture containing purified amplified DNA
Other Reagents, Equipment, and Materials
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Micropipettes and Tips (appropriate volumes)
FastDigest enzymes (to be assigned)
10X FastDigest buffer
1.5 ml microfuge tubes
Nuclease-free water
6X DNA loading dye (Amresco)
ThermoIEC Micromax RF centrifuge (or suitable microcentrifuge)
Procedure:
1)
Using the following formula, prepare a master mix in a 1.5 ml microfuge tube for n + 1
reactions where n = the number of different restriction enzymes that will be used:
Nuclease-free water
(n + 1) x 7 µl
10X FastDigest buffer
(n + 1) x 2 µl
PCR DNA
(n + 1) x 10 µl
2)
To n 1.5 ml microfuge tubes, pipette 19 µl of the master mix.
3)
To each microfuge tube, add 1 µl of an appropriate restriction endonuclease.
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BIOL 3716 Protocol: Restriction endonuclease analysis of PCR products
v. 091508
4)
Mix each reaction solution by gently flicking the microfuge tubes.
5)
Collect the solutions by centrifugation for 10 sec at room temperature (approximately 25°C)
using a microcentrifuge operating at 10,000 rpm.
6)
Incubate the microcentrifuge tubes at 37°C for 20-25 minutes.
7)
To each microfuge tube, add 4 µl of 6X DNA loading dye and mix by gently flicking the
microfuge tubes.
8)
Collect the solutions by centrifugation for 10 sec at room temperature (approximately 25°C)
using a microcentrifuge operating at 10,000 rpm.
9)
Resolve the restriction endonuclease products by electrophoresis using a 1.5% agarose gel
(follow the procedures outlined in Protocol: Gel Analysis of PCR Products).
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