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* [http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechPCR.shtml Polymerase Chain Reaction (PCR) at NCBI] | * [http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechPCR.shtml Polymerase Chain Reaction (PCR) at NCBI] | ||
=== | === Polymerase chain reaction (PCR) protocol === | ||
* | * Assemble PCR mixture | ||
** PCR | ** PCR master mix - 45.0 μL | ||
*** Nuclease-free | *** Nuclease-free water -35.6 μL | ||
*** 10X PCR | *** 10X PCR buffer - 5.0 μL | ||
*** Forward | *** Forward primer (VF2) - 1.6 μL | ||
*** Reverse | *** Reverse primer (VR) - 1.3 μL | ||
*** Deoxynucleotide | *** Deoxynucleotide triphosphate (dNTP) mix - 1.0 μL | ||
*** | *** ''Taq'' DNA polymerase - 0.5 μL | ||
** Template DNA ( | ** 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/m0273-taq-dna-polymerase-with-standard-taq-buffer | * [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 | * [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]] | |||
</div> | </div> | ||
Latest revision as of 13:51, 18 March 2013
Home Contact Internal Lab Members Projects Protocols Build-a-BUG Build-a-Gene 2016 Build-a-Gene 2015 Build-a-Gene 2014 Build-a-Gene 2013 Chalk Talks
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
- PCR master mix - 45.0 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
