Transcript pARA-R Restriction Digest: An Introduction to Plasmids and

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pARA-R Restriction Digest:
An Introduction to Plasmids and Restriction Enzymes
Laboratory 2a
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Overview
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Purpose:
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Examine role of restriction endonucleses (enzymes) in genetic
engineering
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Examine a bacterial plasmid and its use in biotechnology
Methods:
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Preparing the pARA – R restriction digest
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Introduction
Restriction enzymes
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Cut DNA molecules from various
organisms and recombine pieces
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Recombinant DNA
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Restrict the growth of viruses in
bacteria
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Digest the DNA molecule at specific
nucleotide sequences
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Restriction fragments
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DNA fragments
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Sticky ends
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Allow annealing and
recombination of DNA fragments
from different sources
Engineering the Plasmid: ligation of rfp gene into p-ARA
Bruce Wallace
BamH I
sticky end
Hind III
sticky end
Hind III
sticky end
BamH I
sticky end
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Introduction
Bacterial plasmids
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Circular pieces nonessential DNA
found in bacteria
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Can be engineered to carry genes
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Express proteins encoded by these
genes
pARA – R
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Recombinant DNA plasmid
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Engineered to express rfp gene
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Produces a mutant Red
Fluorescent Protein (mFP)
Restriction digest of pARA-R
Recombinant plasmid of interest
pARA-R
4720 bp
rfp
702bp
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Recombinant Plasmid pARA - R
Important control elements:
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araC
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Protein to help bacteria make other
proteins
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Encoded by genes inserted into
plasmid
pBAD
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Site where RNA polymerase binds to
initiate transcription
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rfp
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Hind III & BamH I
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Restriction enzymes
ampr
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Antibiotic resistance gene
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Encodes beta lactamase
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Materials
Reagents:
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Equipment & Supplies
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pARA-R (70 ng / μL)
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P-20 micropipette and tips
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Restriction enzymes
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1.5 mL microfuge tubes
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BamH I
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Minicentrifuge
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Hind III
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37oC water bath
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Markers
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2.5x restriction buffer
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Distilled water (dH2O)
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What will you need to do?
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Aliquot:
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pARA – R
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Enzyme mix
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2.5x restriction buffer
Turn on water bath the day before lab
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37oC
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Methods
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1. Preparing the pARA-R
Restriction Digest
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Three tubes:
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pARA-R
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Enzyme mix
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2.5x restriction buffer
Tube
2.5x
buffer
dH2O
pARA - R
Enzyme
mix
Total
volume
A+
4 μL
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4 μL
2 μL
10 μL
A-
4 μL
2 μL
4 μL
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10 μL
Obtain 2 clean 1.5 mL microfuge tubes
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Label as “A+” and “A-”
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Use fresh tip and add 4 μL of 2.5x restriction
buffer to both tubes
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Add 2 μL dH2O to “A-”
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Fresh tip and add 4 μL of pARA – R to both
tubes
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1. Preparing the pARA-R
Restriction Digest
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A+ tube
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Teacher will dispense enzyme mix
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2 μL
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Cap tubes and gently “flick” to mix
Tube
2.5x
buffer
dH2O
pARA R
Enzyme
mix
Total
volume
A+
4 μL
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4 μL
2 μL
10 μL
A-
4 μL
2 μL
4 μL
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10 μL
Minifuge
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Balance with other tubes
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Place both tubes in 37oC water bath for 60 minutes
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Either go directly to Lab 4a or freeze until ready to complete
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Conclusions
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Will result in 2 restriction fragments
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4018 bp with ampr gene and control regions
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702 bp with rfp gene
Bruce Wallace
Restriction analysis of pARA-R
Restriction fragments after digest with Hind III and BamH I
BamH I
Hind III
4018 bp
BamH I
Hind III
702bp