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18 Cards in this Set
- Front
- Back
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Which is more expensive? Quiagene or Squish prep?
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Quiagene @ 5$ per sample
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What organism did we use?
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Drosophila melanogaster
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What ingredients do we use?
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Water, 5M NaCl, 0.5M EDTA, 1M TRIS-HCl (pH 8.0), Proteinase K (20mg.ml
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What does protienase K do?
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It digesta almost all proteins except itself and is the key component in the squish prep
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How much of each do we put in?
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441.5 uL water
2.5uL of 5M NaCl 1.0uL of EDTA 50uL of 1M Tris-HCl 5uL of Proteinase K Mix by gently flicking the bottom of the tube |
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What comprises of the squish?
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The fruit fly in 5uL of the master mix and an added 45uL of the master mix
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What do you do after prepping the squish?
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Incubate it using a thermocycler for 60 min at 65 deg celcius (optimal for prot K)
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What happens for 5 mins at 95 deg celcius?
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It kills protease K activity
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Why is the lid heated?
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to prevent condensation
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What happens after incubation
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PCR
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Ingredients?
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Water, 10x PCR buffer, 5mM dNTPs, 10uM LCO1490 primer (it flanks the left side of the barcode), 10uM HCO2198 Primer (flanks the right side of the barcode),
5U/uL Taq polymerase. |
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How much of each is needed for 10 experiments?
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Water 199uL
10x PCR buffer 25uL 5mM dNTPs 5.0 uL 19uM LOCO 1490 5uL 10uM HCO2198 5uL 5u/uL taq polymerase 1 uL Squish prep 1uL Add 24uL of PCR master mix to 1uL of squish |
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What happens after making your sample?
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Amplification using Invertebrate barcoding program
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What are the 4 steps in this process?
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Stage 1 is to completely denature DNA
Stage 2 is for pre-amplification (used to increase sensitivity) Stage 3 is for amplification of barcode. Stage 4 is for extension and cooling |
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What step comes next?
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gel electrophoresis.
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What does pouring extra elution buffer do?
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It further submerges the gel
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When loading the samples, what do the dyes do?
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They aid in loading and monitor migration
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Next, you load the PCR sample and remove the gel and put it onto the gel dock to be read. Then you receive a gel image. We expect a band at approx 650 bp. The first 6 markers are at 100bp intervals. Send sample for DNA sequencing
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lul
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