Confirmation of pARA–R Restriction Digest Laboratory 4a

Do Now What is arabinose? What is ampicillin? http://ecoliwiki.net/colipedia/index.php /pBAD/Thio-E

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.

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 1961. 

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- --------

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

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

Video Clips

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

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:

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

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

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

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

Methods

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

Video Clip http://www.teachersdomain.org/asset/biot11_vid_isol atedna/

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

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 30-40 minutes

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

Prediction for restriction gel Restriction analysis of pARA-R Bruce Wallace Prediction for restriction gel M A+ A- M A+ A- 500 1000 1500 2000 3000 4000 5000 8000 10000