1. pARA-R Restriction Digest: An Introduction to Plasmids and Restriction Enzymes
Laboratory 2a

2. Overview Purpose: Methods:
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

3. 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

4. Engineering the Plasmid: ligation of rfp gene into p-ARA
Bruce Wallace
sticky end
BamH I
sticky end
Hind III
sticky end
Hind III
sticky end
BamH I

5. 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)

6. Recombinant plasmid of interest
Restriction digest of pARA-R
Recombinant plasmid of interest
pARA-R
4720 bp
rfp
702bp
BamH I
Hind III

8. 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

9. Materials Reagents: pARA-R (70 ng / μL) Restriction enzymes BamH I
Hind III
2.5x restriction buffer
Distilled water (dH2O)
Equipment & Supplies
P-20 micropipette and tips
1.5 mL microfuge tubes
Minicentrifuge
37oC water bath
Markers

10. What will you need to do? Aliquot:
pARA – R
Enzyme mix
2.5x restriction buffer
Turn on water bath the day before lab
37oC

11. Methods

12. 1. Preparing the pARA-R Restriction Digest
Tube
2.5x buffer
dH2O
pARA - R
Enzyme mix
Total volume A+
4 μL
2 μL
10 μL A-
Three tubes:
pARA-R
Enzyme mix
2.5x restriction buffer
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
Double these numbers!! =
20μL

13. 1. Preparing the pARA-R Restriction Digest
A+ tube
Teacher will dispense enzyme mix
2 μL
Cap tubes and gently “flick” to mix
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
Tube
2.5x buffer
dH2O
pARA - R
Enzyme mix
Total volume A+
4 μL
2 μL
10 μL A-
Double these numbers!! =
20μL

14. Conclusions Will result in 2 restriction fragments
4018 bp with ampr gene and control regions
702 bp with rfp gene

15. Restriction fragments after digest with Hind III and BamH I
Restriction analysis of pARA-R
Bruce Wallace
Restriction fragments after digest with Hind III and BamH I
BamH I
Hind III
4018 bp
Hind III
BamH I
702bp