1. Rec. DNA Lab Week 2 Restriction Digestion and Agarose Gel Electrophoresis

2. Overview –Assay our Plasmid preps from last week by digesting a portion of the prep with restriction endonucleases. –These digests will then be run on an agarose electrophoresis gel and stained with Fast Blast This Lab (Ex. 4) and Thursday’s lab (Ex. 6) will be combined into one write-up

3. Restriction Endonucleases Enzymes that recognize specific sequences of DNA and cleave the sugar backbone Originally named for the restriction of phage growth in certain bacterial strains –Methylases/nucleases –1968 H.O. Smith, K.W. Wilcox, and T.J. Kelley isolated and characterized the first sequence specific restriction nuclease

4. Restriction Endonucleases Type II - Cuts at or near a short recognition sequence. Separate enzyme methylates the same recognition sequence. –Palindromic vs. non-palindromic rec.sites –Blunt vs. Sticky ends –Type I - Cuts nonspecifically and far from recognition sequence. Contains both restriction and methylation activities. –Type III - Cuts 24-26 bp downstream from a short, asymmetrical recognition sequence. Requires ATP and contains both restriction and methylation activities

5. Using Restriction Endonucleases Keep cold! –Enzymes loose activity at room temperature Pipette from the surface of the liquid –Dipping into the enzyme gets too much on the tip Different restriction endonucleases use different buffers- make sure you use the right one –Buffers are usually 10X, add 1 ul for each 9 ul of total reaction volume

6. Agarose Gel Electrophoresis A gel matrix that uses an electrical current to separate molecules based on mass/charge rations –Because DNA fragments have the same charge, they are separated by mass –Larger fragments move slower, smaller move faster

7. Using our gel rigs : Cool agarose for several minutes before pouring “Caulk” the edges of casting tray before filling Be careful removing and moving gels, they are slippery and fragile

8. Fast Blast Staining Procedure Wearing gloves, remove the gels from their trays and submerge them in the 100x stain in an appropriately sized container. Stain the gels for 2–3 min, but not more than 3 min. Save the used 100x stain for future use. The stain can be reused at least 7 times. Transfer the gels to a large container containing 500– 700 ml of clean warm (40–55°C) tap water. Gently shake the gels in the water for ~10 sec to rinse. Transfer the gels to a large container with 500–700 ml of clean warm tap water for 5 min Move the gels gently in the water once every minute. Perform a second wash in clean warm water as in previous step

9. Changes to the lab: 1.pBR322 will not be used, substitute your purified plasmid instead - pGEM 2.Use 150 mL of 0.8% agarose instead of 80 mL as the book says 3.Use the buffer that is specific for each restriction endonuclease 4.Endonuclease NdeI will replace PstI 5.Fast Blast stain after electrophoresis instead of Et-Br

10. Checklist Per Table: –Set up tubes A-F and digest with restriction enzymes Remember to save tube B…You will need this in future labs! –Run gel of digests and photograph Photographs will be available for download from Web CT after class –Combine Ex 4 and 6 in one lab report Include answers to questions 1-8 in Ex. 4 –Thursday’s lab: review gels and prepare media for use in future labs Environmental Isolation – Round II Introduce genomic DNA purification protocol – handout Turn in Exercise I Write-up