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