PCR Procedure<\/p>\n
Materials:
\nDNA Extractions
\nForward and reverse primers
\nPremix taq from freezer
\nEmpty Eppendorf tube
\nEppendorf tube of PCR water
\nPositive control (Specimen already known to be high quality)
\nTwo 96-well plates
\nEppendorf vial rack<\/p>\n
Preliminary Procedure:
\n\u2022\tPrimers need to be diluted from 10 micromolar to 1 micromolar to be stocked in the freezer for 1-2 months.
\n\u2022\tPut your sample information in the database to make new sample numbers for the PCR.
\n\u2022\tLabel the strip tubes with the new sample numbers using an alcohol resistant marker and put them into a PCR plate. Add extra labels at the end for positive and negative controls.
\n\u2022\tLook in \u201cDNA methods\u201d section of lab website for cycling methodology.
\n\u2022\tLook in the database to find primers in the freezer.
\n\u2022\tUse the PCR excel chart to determine the amount of reagents you need to use in the master mix (the final volume of the master mix should equal 25 uL).
\n\u2022\tUV the PCR hood for 15 minutes before starting to do the PCR. <\/p>\n
Master Mix Procedure:
\no\tAdd x uL of Taq to the empty Eppendorf tube
\no\tAdd x uL of each primer to the tube, pipetting up and down to mix
\no\tAdd x uL of PCR water to the tube
\no\tVortex the master mix for 5 seconds<\/p>\n
PCR Procedure:
\n\u2022\tVortex the extractions for 1 second, and then centrifuge to eliminate any bubbles.
\n\u2022\tMake sure that the DNA extractions are in the same order that the empty PCR tubes are in.
\n\u2022\tPipette 23 or 24 uL of master mix into each PCR tube using the same tip.
\n\u2022\tPipette 1 or 2 uL of each DNA extraction into the PCR tubes. Use a different pipette tip each time, and mix the liquid up and down with the pipette. Try not to introduce bubbles. Close each cap when done pipetting so that none are skipped or none have double the correct amount added.
\n\u2022\tAdd 1 or 2 uL of PCR water only to the negative control tube.
\n\u2022\tClose the tops of all the vials and make sure that all of them are closed. Sometimes they do not close all the way even if they look closed.
\n\u2022\tTap the plate to the table to bring drops in the tubes down.
\n\u2022\tCentrifuge strip tubes for 3 seconds to make sure there are no bubbles.
\n\u2022\tPut the tubes into the smaller holes of the thermocycler. Make sure the caps are completely closed. Add other tubes of the same kind to balance it.
\n\u2022\tRun the program called for in the PCR protocol, and set a timer to be able to take the PCR products out and put them in the freezer.
\n\u2022\tPut the extractions and any unused reagents back in the freezer in your box.
\n\u2022\tThe PCR products should be used as soon as possible.
\n\u2022\tClean everything with ethanol and UV the hood.
\n .
\n .
\n .
\n .
\nEnsure you enter all PCR runs into the PCR form on the isolations database. You can use the form linked here to properly plan your PCR master mix and database entries. Use the database entry rows at the top to fill in your samples for one run, including a positive control (known working template for these conditions), an extraction negative control (negative control from extraction as template), and a PCR negative control (no template). Then put in the number of reactions in the master mix form, this will give you the necessary amount of reagents to make master mix for your reactions plus two additional.<\/p>\n
It is recommended you input your PCR database entries before beginning mixing and your PCR run, this will reserve the spots so that the PCR IDs do not get taken while you are working.<\/p>\n