446/546 handout 31 Aug. PCR AND homework.
GENERAL ADVICE,
You will be pipetting small volumes of expensive reagents into small tubes. Read your instructions
carefully and ASK when in doubt. Use clean tips at all time, if you are not sure, take another clean
tip. Keep stock reagents on ice as much as possible. Wear gloves, coat when handling chemicals.
Gloves also protect the samples FROM YOU, reducing the potential of contamination of your PCR
reactions. Background, manuals for the PCR reagents will be available on-line
Needed: parasite and snail genomic DNA samples, special PCR tubes, AmpliTaq Gold, 10x Gold
buffer, MgCl2, dNTPs, mQ water
Every group: 4 PCR primer sets, mitochondrial rDNA 16S, mitochondrial CO1, rDNA 18S, 28S
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Snail and parasite PCR (Hot start, regular PCR)
1) Make sure that all reagents are completely thawed before you pipet. Make a master mix
(combination of all common reagents) in a 1.7 ml snap cap. Order is important. Spin tubes
briefly to collect volume at the bottom.
EACH GROUP MAKES ONE MASTERMIX,
66l mQ water
20 l 10X buffer
32 l MgCl2
32 l dNTPs
2 l TaqGold (look to see it descend to the bottom)
TOTAL VOLUME is 152 l
MIX, spin down.
2) Dilute your genomic DNA sample from the snail 1/50: combine 5 l with 245 l mQ water.
Label this tube clearly for identification and place it in your freezer box AFTER use.
3) Label the lid of 4 PCR tubes with group number and reaction number (e.g. 1.1; 1.2; 1.3; 1.4),
place 38 l of the master mix in each of these tubes
4) To reaction tube number 1 and 2
add 8 l mQ water and 2 l of the 1/50 diluted genomic DNA sample from the SNAIL.
5) Pipet the following primers under the surface of the reaction volume
reaction 1: add 2 l of primers for 16S (these are primers PU178 and PL1462),
reaction 2: add 2 l of primers for CO1 (primers SCO1HCO2198 and SCO1LC1490).
6) To reaction tube number 3 and 4
add 10 l of the genomic DNA sample from the PARASITE.
7) Pipet the following primers under the surface of the reaction volume
reaction 3 add 2 l of primers for 18S (these are primers worm A and worm B),
reaction 4: add 2 l of primers for 28S (primers primers LSU and 1500R).
(continued other side)
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8) Close the caps on your reaction tubes, SPIN DOWN, and give them to an instructor to run the
PCR thermal cycling.
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Homework, answer in your note book
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Describe the three phases of regular PCR thermal cycling.
How many different primers are needed for a PCR reaction to work?
What is the Tm of a PCR primer?
Primer extension during a PCR reaction is directional.
Does the reaction proceed 5’ -> 3’ or 3’ -> 5’?
What are benefits from making a mastermix?
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