PCR

Lab 2: Polymerase Chain Reaction Purpose We will use Polymerase Chain Reaction to amplify the GFP gene. Using a DNA Polymerase called "Taq" and two primers that complement the two ends of the GFP DNA, we will create a huge number of linear copies of GFP DNA. In the next lab this DNA will be inserted into an antibiotic resistant vector. Materials Equipment 2-20μL Air displacement pipette (1 per 2 students) • Pipette Tips • 0.2mL (PCR) Tube • Thermal Cycler (1 per class) Reagents Methods You will be mixing together the appropriate reagents in given amounts, just as you practiced in the first laboratory session. Use the table on the right below to reference how much of each reagent, adding each reagent in from top to bottom in the order listed. It is important to remember good pipetting skills to ensure that there is enough of each reagent for everyone in your group.
 * Reagents from your group’s bag 5x PCR Buffer
 * DNTPs
 * GFP Forward Primer
 * GFP Reverse Primer
 * DNA Template
 * • Taq Polymerase

Amount

5x PCR Buffer 4.0μL DNTPs 5.0μL GFP For. 3.0μL GFP Rev. 3.0μL DNA Template 4.0μL Taq Polymerase 1.0μL Total 20.0μL

1.**Label ** your PCR tube on the cap and the side with your initials using a dark permanent marker. 2.**Add the PCR reagents to the tube ** . Wait for all reagents to thaw completely before pipetting. a. Add 4.0μL of 5x PCR Buffer to the tube. b. Add 5.0μL of DNTPs c. Add 3.0μL of GFP Forward Primer d. Add 3.0μL of GFP Reverse Primer e. Add 4.0μL of DNA Template f. Add 1.0μL of Taq Polymerase g. Mix Tube gently. 3. Close the tube and bring it to the Thermal Cycler. Place the tube in an open tube slot. Make sure your tube is properly labeled. Once all the students in the class are finished putting together their reactions. The instructor should start the PCR machine on the "GFP_PCR" program (the details of this program are found in the CAFG Instructor’s Manual). Clean-up PCR Tubes may remain in the Thermal Cycler overnight, but for longer periods, they should be stored in the refrigerator. Unused reagents may be thrown out unless your instructor is collecting them. Double check to make sure that your PCR Tube is labeled with your initials so that it does not become mixed up with other tubes. Conclusions By the end of the thermal cycling your PCR Tube should contain a large number of copies of short sequences of DNA that encode for only the GFP gene. During the next lab period you will be inserting this short DNA sequence into a larger piece of circular DNA that can then be put into bacteria for expression. []