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pARA-R Restriction Digest: An Introduction to Plasmids and Restriction Enzymes
Laboratory 2a
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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
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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
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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
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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)
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Recombinant plasmid of interest
Restriction digest of pARA-R Recombinant plasmid of interest pARA-R 4720 bp rfp 702bp BamH I Hind III
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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
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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
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What will you need to do? Aliquot:
pARA – R Enzyme mix 2.5x restriction buffer Turn on water bath the day before lab 37oC
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Methods
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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
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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
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Conclusions Will result in 2 restriction fragments
4018 bp with ampr gene and control regions 702 bp with rfp gene
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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
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