Date: 3/22/18

Purpose: To conduct PCR reactions using the two different DNA extraction protocols from last week and create the agarose gel.

Procedure:

• Calculate the volumes needed (10mL of 10x TAE stock solution and 90mL of DI water)
• Label tubes 1-6 (tubes 1-3 are COX1 primer and tubes 4-6 areV4 primer)
• Add the proper amounts of solutions to each tube
• Tube 1: 12.5ul master mix, 0ul DNA, 0.6ul 20uM COX1/11.9ul water
• Tube 2: 12.5ul master mix, 1ul DNA, 0.6 20uM COX1/10.9ul water
• Tube 3: 12.5ul master mix, 1ul eDNA, 0.6 20uM COX1/10.9ul water
• Tube 4: 12.5ul master mix, 0ul DNA, 0.6 20uM V4/11.9ul water
• Tube 5: 12.5ul master mix, 1ul DNA, 0.6 20uM V4/10.9ul water
• Tube 6: 12.5ul master mix, 1ul DNA, 0.6 20uM V4/10.9ul water

Label initials on tubes and place in front of classroom in tube racks

Preparing 1.8% agarose gel

• Make 100mL 1x TAE stock solution using 10mL of 10x TAE stock solution and 90mL of DI water
• Transfer 35mL of the solution to new flask and add in 0.6g of agarose
• Lightly place lid on flaskHheat flask in microwave at power 7 for 1 min and 20 sec
• Let flask cool for about 5 min
• Set up agarose gel box and comb
• Have instructor add in 2ul of ethidium bromide to the gel and mix using a swirling motion
• Carefully pour gel into the gel box with the comb already inserted in
• Fill out QTM and the protocol puzzle

Tubes were labeled with a star (*)

Conclusion:

The purpose of today’s lab was to prepare the PCR reaction for the extracted DNA from the Chelex or PowerSoil protocol depending on which one the group conducted. Our group used the Chelex extraction protocol. As a result of conducting the PCR reaction before for the Ludox protocol, students were familiar with the process and were more experienced while conducting it again.  Since the previous time we conducted PCR did not give us the desired nor any results, we hope that this trial brings some results to analyze and improve on for next time. For next week’s lab we will be conducting gel electrophoresis.