2010 Bio 311 Lab B Manual (part 2)

2010 Bio 311 Lab B Manual (part 2) - B3. Cleanup of...

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57 B3. Clean–up of Digested DNA Lab Overview: In order to subclone EGFP, you must ligate the EGFP fragment to the prepared pET28b vector. You first need to isolate the insert DNA through agarose gel electrophoresis, and then clean it and the vector DNA sample up using a glassmilk cartridge so that you can add vector and insert together in a buffer compatible for ligation. Isolation of the EGFP insert DNA using agarose gel electrophoresis A. Agarose Gel Electrophoresis Materials power supply Agarose (not agar ) 10X TAE buffer (40 mM Tris-acetate, 1mM EDTA) Sample IBE = pEGFP cut with restriction enzymes ( IBE) Lambda Bst EII ( λ ) SYBR green (10,000X stock) (green tube) Horizontal electrophoresis tank with casting tray, 1-10 teeth comb, and lid 6X TD 1. Procedure for agarose gel: a. Prepare a 1% agarose gel and 1X TAE buffer as described in Lab B2. (Don’t forget to add the SYBR green.) b. Set up the agarose gel s described in Lab B2 c. Measure the left over volume of the sample IBE (insert cut with Eco RI and Bam HI). Divide the volume by 5 and add that much 6X TD to the sample to prepare it for gel electrophoresis. (eg, if the volume is 10 μ l, then add 2 μ l of 6X TD for a total of 12 μ l.) If you did not see 2 bands on the gel for your IBE digest, ask your TA for an additional IBE sample to run on the gel in lane 4 or 5. d. Load your samples: consolidate IBE into a few wells as possible 9 Well Sample ± 1 Lambda Bst EII standard ( λ ) ± 2 leave empty ± 3 fill the well with IBE (pEGFP cut with Bam HI & Eco RI) ± 4 if need be put the remainder of IBE
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58 2. Slide the top onto the electrophoresis tank with the leads connected properly, red to red, and black to black. 33 ALWAYS watch the gel for the first 5 minutes of the run to ensure that the DNA is migrating in the right direction. 3. Switch to the low setting on the power supply. Turn on the power and turn the knob until the display reads 140 Volts. 4. While you wait, weigh a 1.5 ml microcentrifuge tube and record its weight. Mark it “I” 5. Run the gel until the bromophenol blue dye travels about an inch from the bottom Turn off the power. G Always turn off the power before disconnecting the leads and removing the lid. B. Extracting the insert DNA fragment from the gel 1. G Wearing gloves remove your gel from the casting tray and place on the plastic box in the prep room (042A). With the lights out, shine a black light over the gel and locate the smaller piece of insert DNA in lanes 3-4. Clean your spatula well with water and then ethanol. With the clean spatula, cut as close as possible into the gel to isolate the DNA for purification. 2. Transfer your piece into a clean microcentrifuge tube that had been weighed while empty. 3.
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2010 Bio 311 Lab B Manual (part 2) - B3. Cleanup of...

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