1. Confirmation of pARA–R Restriction Digest
Laboratory 4a

2. Do Now What is arabinose? What is ampicillin?
/pBAD/Thio-E

3. araC
The L-arabinose operon of the model bacterium E coli has been a focus for research in molecular biology for over 40 years.
It is controlled by a dual positive and negative system. They encode the metabolic enzymes for breaking down the monosaccharide sugar arabinose into D-xylulose- 5-phosphate, which is then metabolised via the pentose phosphate pathway.

4. pBAD
Since the first report of its use as an inducible expression system, the arabinose-inducible araBAD promoter (P-BAD) and regulator (AraC) of Escherichia coli have become popular for controlled gene expression in E. coli and other bacteria (Guzman et al., 1995)
Ampicillin is an antibiotic that has been used extensively to treat bacterial infections since  

5. 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-

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

7. 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

8. Video Clips

9. Objectives
Examine the restriction fragments that result from the double digestion of pARA – R
By BamH I and Hind III
Perform gel electrophoresis to visualize DNA plasmids

10. Introduction DNA fragments move at different speeds
Smaller = faster
Larger = slower
Take digested and undigested plasmid samples
Undigested = control (A-)
2-3 bands may appear
Plasmids isolated form cells may exist in several forms:

11. Introduction Nicked-circle Break in sugar-phosphate backbone
Moves slower than supercoiled
Multimer
Replicated plasmids that remain linked together
Move the slowest
Supercoiled
Compact molecule
Move quickly through gel

12. Different Structural Forms
circle
“multimer”
Nicked Circle
Linear
Supercoiled
“nicked-circle”
Different structural forms produce different bands.

13. Materials Reagents Plasmid samples 0.8% agarose gel 5 x loading dye
1 x SB
DNA size marker (25 ng/μL)
Equipment & Supplies
P-20 micropipette and tips
1.5 mL microfuge tubes
Electrophoresis equipment
Power supply
Plastic microfuge tube rack
Markers
Trans-illuminator
Camera set-up
Tube floats
Staining trays

14. What will you need to do? Agarose gel Aliquot:
10 μL of 1 Kb ladder (marker) for each group

15. Methods

16. Do Now
What will the electophoresis gel shows us about restriction enzymes?
What do you think about our time with biotechnology?

17. Video Clip
atedna/

18. Methods 3 tubes Add 2 μL loading dye to tubes A- and A+
DNA marker
Add 2 μL loading dye to tubes A- and A+
Loading dye increases density so DNA will sink
Pump pipette to mix samples
New tip for each plasmid
Prepare the gel
Load samples

19. Methods Loading samples: Connect leads and turn on
Use clean tip and pipette 10 μL of “DNA size marker”
Dispense as directed in Lab 1
Using clean tip load 12 μL A- into adjacent well
Using clean tip load 12 μL A+ into adjacent well
Connect leads and turn on
As directed in Lab 1
Let run about minutes

20. Conclusions pARA–R Should see 2 – 3 DNA bands 3 bands in A-
Has enzymes

21. Prediction for restriction gel
Restriction analysis of pARA-R
Bruce Wallace
Prediction for restriction gel M A+ A- M A+ A-
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