BUGSS:Build-a-BUG:2-3: Difference between revisions

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=== Background reading ===
=== Background reading ===
* Review [[BUGSS:Build-a-BUG:2-1 | Session 1]] and [[BUGSS:Build-a-BUG:2-2 | Session 2]]
* Review [[BUGSS:Build-a-BUG:2-1 | Session 1]] and [[BUGSS:Build-a-BUG:2-2 | Session 2]]
* [http://www.ncbi.nlm.nih.gov/pubmed/4377758 DNA ligase: structure, mechanism, and function] (Requires subscription; I will look for an open access alternative)
* [http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechPCR.shtml Polymerase Chain Reaction (PCR) at NCBI]


=== Protocols ===
=== Polymerase chain reaction (PCR) protocol ===
* [https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202 DNA ligation with T4 DNA Ligase]
* Assemble PCR mixture
* [http://carroll.molbio.wisc.edu/methods/Bacterial/CompetentCells.html Making competent ''E. coli''] (BUGSS has done this for you)
** 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 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J63002 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 ===
=== Materials ===
* [https://www.neb.com/products/m0202-t4-dna-ligase T4 DNA Ligase]
* [https://www.neb.com/products/m0273-taq-dna-polymerase-with-standard-taq-buffer ''Taq'' DNA polymerase]
* [http://www.promega.com/products/pcr/routine-pcr/dntp-mix/ Deoxynucleotide triphosphate (dNTP) mix]
 
== 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 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J63002 BBa_J63002]).
 
=== Gel Images ===
[[Image:BBa_J63002_Digest_Gel.jpg|thumb|left|760px|alt=BBa_J63002 Digest Gel|BBa_J63002 Digest Gel]]
[[Image:Deb's_PCR_Product_Gel.jpg|thumb|left|760px|alt=Deb's PCR Product Gel.jpg|Deb's PCR Product Gel.jpg]]


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Latest revision as of 13:51, 18 March 2013

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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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