BUGSS:Build-a-BUG:2-3

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Current revision (15:51, 18 March 2013) (view source)
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*** Forward primer (VF2) - 1.6 μL
*** Forward primer (VF2) - 1.6 μL
*** Reverse primer (VR) - 1.3 μL
*** Reverse primer (VR) - 1.3 μL
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*** Deoxynucleotide Triphosphate (dNTP) Mix - 1.0 μL
+
*** Deoxynucleotide triphosphate (dNTP) mix - 1.0 μL
*** ''Taq'' DNA polymerase - 0.5 μL
*** ''Taq'' DNA polymerase - 0.5 μL
** Template DNA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J63002 BBa_J63002]) - 5.0 μL
** Template DNA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J63002 BBa_J63002]) - 5.0 μL
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** Extend at 72°C for ? sec
** Extend at 72°C for ? sec
** Repeat 29 times (for 30 cycles total)
** Repeat 29 times (for 30 cycles total)
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** Cool to 4°C
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** Hold at 4°C
=== Materials ===
=== Materials ===

Current revision

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Contents

Build-a-BUG: Series 2, Session 3

Saturday, March 9, 2013
Noon to 4:00 PM

This is the third Build-A-BUG workshop in a series of five on yeast. You will learn more about molecular cloning and continue to hone your basic lab techniques, while continuing either your own or somebody else's mating type detector project.

Background reading

Polymerase chain reaction (PCR) protocol

  • Assemble PCR mixture
    • PCR master mix - 45.0 μL
      • Nuclease-free water -35.6 μL
      • 10X PCR buffer - 5.0 μL
      • Forward primer (VF2) - 1.6 μL
      • Reverse primer (VR) - 1.3 μL
      • Deoxynucleotide triphosphate (dNTP) mix - 1.0 μL
      • Taq DNA polymerase - 0.5 μL
    • Template DNA (BBa_J63002) - 5.0 μL
  • Amplify in thermal cycler
    • Melt at 95°C for ? sec
    • Anneal at ?°C for ? sec
    • Extend at 72°C for ? sec
    • Repeat 29 times (for 30 cycles total)
    • Hold at 4°C

Materials

Notes

  • We planned to ligate all parts and transform E. coli during this session. Unfortunately, we ran into two problems: the alpha-cell promoter part was not ready and the ADH1 terminator did not cut as expected (see gel image below).
  • After weighing all options, we decided to PCR amplify the ADH1 terminator (BBa_J63002).

Gel Images

BBa_J63002 Digest Gel
BBa_J63002 Digest Gel
Deb's PCR Product Gel.jpg
Deb's PCR Product Gel.jpg
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