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8:51AM Tue Mar 26 ull T () 76% @B Section 3: PCR setup Steps 1 and 2 of this section will be performed as a lab group, but all other steps will be performed either individually or in pairs. To maximize our chances for success we will be using your group’s cDNA from the previous week as well as the cDNA from each of the other groups. 1. Asalab group label 3 clean 0.65 ml tubes with the number of your lab group. 2. Retrieve your cDNA from last week's lab and transfer 10ul of the solution to each of the labeled tubes. Give each of the labeled tubes to another lab group. Use the charts on the next page as you set up your PCR reaction. You will be making a master mix with all of the components for the PCR and then putting 25pl of this mix into each PCR tube. After the master mix has been divided between each tube you will then put a sample of the various cDNAs into the appropriate tube. 5. Write the Region| Primers 1 2 3 4 \5 name of — your gene in the [N - € r TL -% -C \ column = labeled A "region" and the |EN- K names of - our forward and reverse primers in the "primers" column. ] 2 3 a 5 4. Write the symbol for your lab group in column H.O 20 40 60 80 10n one and enter the symbols for the other MaCl, 075 | 1.5 | 2.25 3 575 cDNAss in the following columns. 10X 2.5 5 & 10 | 2+ 5. Once you have all the cDNA symbols filled in DNTP 0.5 1 1.5 2 2.5 write "water" in the next open column and ~ F Primer 0.5 1 1.5 2 2.5 count the total number of columns used. RPOmSF 0.5 L 1.5 2 2.5 ATaq 0.75 | 1.50 | 2.25 |{ 3.00/) 3.7~ 6. Locate the corresponding number in the lower chart and circle the column below that number. This gives you the volume in pl {8F your mastermix. Get a strip of PCR tubes and caps from your instructor. Set the strip of PCR tubes in a rack. 9. In the tube on the right of your strip carefully add the appropriate amount of each listed solution in your circled column to create your master mix. Make sure you measure carefully and use a new tip for each solution. 10. Once your master mix is done set a pipette to 25ul, mix well by pipetting up and down several times, and transfer 25ul of mix to each empty PCR tube. If you have measured correctly this should leave you with slightly more than 25ul of the mix left in the tube you used to make your master mix. 11. Add 1pl of the cDNA listed in each column to the corresponding tube. Do not add any DNA to thetube in the column labeled "water." 12. Cap the PCR tubes securely and label the side of tube number one with your initials and lab group number. 13. Give your PCR tubes to your instructor for placement in the thermocycler. The solutions will be run under the following cycling profile: (95°C, 30 s.; 50°C, 30 s.; 72°C, 45 s.) x30 14. Clean up and go home. el Q4.4 Why did you do one reaction without any DNA in it? _ ® -
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