Difference between revisions of "DNA sequencing"
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Updated information on the PCR master mix I use and where to get it
(Added more sequencing information)
(Updated information on the PCR master mix I use and where to get it)
Papers on the NaOH extraction:
[[File:Osmundson et al. - 2013 - Back to basics an evaluation of NaOH and alternat.pdf]]
== Polymerase Chain Reaction (PCR) ==
[http://tmcalculator.neb.com Annealing temperature calculator]
I use a 25 μL [https://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] reaction which includes 1 μL of 10 μM forward [https://en.wikipedia.org/wiki/Primer_(molecular_biology) primer], 1 μL of 10 μM reverse primer, 1 μL DNA template and the [https://www.google.com/search?q=pcr+master+mix&oq=PCR+master+mix PCR master mix]. I use the
The current PCR program I am using is an initial denaturation of two minutes at 95 degrees C, followed by 30 cycles of denaturation at 95 degrees for 30 seconds, annealing at 54 degrees for 30 seconds and an extension phase of 72 degrees for 55 seconds. Since the DNA we are amplifying typically isn't very long, it is probably ok to omit the final extension phase of 6 minutes at 70 degrees. The 54 degree annealing temperature was chosen by looking up the [http://biotools.nubic.northwestern.edu/OligoCalc.html melting point] of the primers and subtracting 3 - 5 degrees.
* PCR machine [https://www.ebay.com/sch/i.html?_nkw=PCR+machine $349] (avoid the cheap Thermo Hybaid brand, Josiah says these break really easily. Good ones are MJ Research (usually about $400) and Applied Biosystems (reliable and cheap, like $100, but huge - shipping is like $75)
* 10 uL and 200 uL pipettes [http://www.ebay.com/itm/NEW-Oxford-10-50uL-Adjustable-Micro-Pipette-Series-3000-Sampler-System-Pipet-/291224844297?hash=item43ce5a7809:g:lQIAAOSwxCxT96LG 10 - 50 uL $20] [http://www.ebay.com/itm/Brand-New-Single-Channel-Adjustable-Pipetman-20-200uL-p200-pipet-Pipette-j2e-/182032059910?hash=item2a61f4ce06:g:40QAAOSwiylW~eU2 P200 $22]
* PCR master mix [https
* Electrophoresis DC power supply [https://www.ebay.com/sch/i.html?_nkw=Electrophoresis+power+supply $50]
* Pipette tips (sterile, for p10 and p200 pipettes) $40