components that can interfere with “downstream” protocols. After clean up, our PCR products will be quantitated using spectrophotometry (Nano-Drop instrument). An accurate knowledge of the amount of PCR product is also important for many downstream applications. Materials Buffer PB Buffer PE Elution Buffer EB Spin Column 2 ml collection tube (round botton) 1.5 ml microcentrifuge tubes (conical bottom) (2) PCR Product Clean-up Procedure Before you start: -Be sure appropriate buffers have had ethanol added (marked on bottles) -All centrifugation steps should be performed at 13,000 rpm 1. Transfer entire remaining PCR reaction to a microcentrifuge tube. 2. Add 5 volumes of Buffer PB to your PCR sample and mix. (e.g. add 200 ul of Buffer to remaining 40 ul of PCR reaction). 3. Place a spin column in a provided 2 ml collection tube. 4. To bind DNA, apply the sample to the spin column and centrifuge for 60 s. 5. Discard flow-through. Place the spin column back into the same tube. 6. To wash, add 0.75 ml Buffer PE to the spin column and centrifuge for 60 s. 7. Discard flow-through and place the spin column back in the same tube. 8. Centrifuge the column for an additional 1 min. (IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.) 9. Place spin column in a sterile (DNAse-free) 1.5 ml microcentrifuge tube. 10. To elute DNA, add 50 ul Buffer EB (10 mM Tris·Cl, pH 8.5) to the center of the spin column membrane. Let the column stand for 60 s and then centrifuge for 60 s. (IMPORTANT: Ensure that the elution buffer is dispensed directly onto the spin column membrane for complete elution of bound DNA. 11. Go with your instructor to measure the concentration of your PCR product DNA using the Nano-Drop spectrophotometer. Be sure to record your value in your laboratory notebook. When finished, your instructor will collect your samples for use next week (DNA ligation and Transformation).
4 Assignment – Preparation of a formatted gel electrophoresis figure Prepare a formatted figure of your gel electrophoresis results. This should be done electronically using any number of software programs (Word, Powerpoint, Photoshop, Illustrator, etc.). The figure should be publication grade quality, which includes a figure legend containing all the information usually requested by scientific journals. An example from one of Dr. Larkin’s publications is included below to serve as a model. Figure 1. Temperature response in reverse transcriptase PCR results. Samples: 1) Lemont, 2) Panda, 3) Rexmont, 4) Toro-2, 5) Nato. Temperature (degrees Celsius) of endosperm development is indicated above individual lanes. Molecular weight markers indicated in lane 1 and between samples 4 and 5.
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