BUGSS:Build-a-BUG:2-3

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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
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** Template DNA (3002) - 5.0 μL
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** Template DNA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J63002 BBa_J63002]) - 5.0 μL
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* Amplify in thermal cycler (30 cycles)
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* Amplify in thermal cycler
** Melt at 95°C for ? sec
** Melt at 95°C for ? sec
** Anneal at ?°C for ? sec
** Anneal at ?°C for ? sec
** Extend at 72°C for ? sec
** Extend at 72°C for ? sec
 +
** Repeat 29 times (for 30 cycles total)
 +
** Cool to 4°C
=== Materials ===
=== Materials ===
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== Notes ==
== Notes ==
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*  
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* 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 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J63002 BBa_J63002]).
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=== Whiteboard ===
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=== Gel ===
[[Image:BAB2-2_Agenda.jpg|thumb|left|760px|alt=Build-a-BUG:2-2 agenda|Build-a-BUG:2-2 agenda]]
[[Image:BAB2-2_Agenda.jpg|thumb|left|760px|alt=Build-a-BUG:2-2 agenda|Build-a-BUG:2-2 agenda]]
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[[Image:BAB2-2_BioBricks.jpg|thumb|left|760px|alt=BioBrick parts with sizes and restriction enzymes|BioBrick parts with sizes and restriction enzymes]]
 
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Revision as of 16:37, 18 March 2013

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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)
    • Cool to 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

Build-a-BUG:2-2 agenda
Build-a-BUG:2-2 agenda
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