TITLE: 2-step Polymerase Chain Reaction (PCR)
AUTHORS: Dana Mozaffari, Laura Kvedarauskaite

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Polymerase Chain Reaction (PCR) is a method of making multiple copies of a DNA sequence, involving repeated reactions with a polymerase enzyme, which comes in different types, such as Taq-polymerase, Phusion or Q5.</div><div class = "text-block">To run a PCR reaction, one needs a DNA template to be amplified, one set of forward and reverse primers, a polymerase enzyme as well as various buffers.</div><div class = "text-block">This protocol describes the procedure to perform a two-step PCR reaction, in which the primers contain an extra non-binding overlap sequence which allows the assembly of a new sequence from a specific region of the template DNA. </div><div class = "text-block">This is done by using two sets of primers, one which adds a non-binding overlap sequence and one other set which add a desired sequence binding to the overlap region introduced by the first set of primers.
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[STEPS]
?. [Gene specific PCR]
Set up the thermocycler programFor initial denaturation98°C for 2 minutesFor 35 cycles:98°C for 30 seconds for denaturation30 seconds for primers annealing at temperature depending on primers and polymerase72°C for 30 seconds per kb of DNA for elongationFinal extension:72°C for 7 minutes4°C on hold
For 35 cycles:98°C for 30 seconds for denaturation30 seconds for primers annealing at temperature depending on primers and polymerase72°C for 30 seconds per kb of DNA for elongationFinal extension:72°C for 7 minutes4°C on hold
Final extension:72°C for 7 minutes4°C on hold
?. [Extension PCR]
Set up the thermocycler programFor initial denaturation98°C for 30 secondsFor 10 cycles:98°C for 8 seconds for denaturation20 seconds for primers annealing at temperature depending on primers and polymerase72°C for 30 seconds per kb of DNA for elongationFinal extension:72°C for 7 minutes4°C on holdFor 10 cycles:98°C for 8 seconds for denaturation20 seconds for primers annealing at temperature depending on primers and polymerase72°C for 30 seconds per kb of DNA for elongationFinal extension:72°C for 7 minutes4°C on holdFinal extension:72°C for 7 minutes4°C on hold
?. [Final PCR]
Set up the thermocycler programFor initial denaturation98°C for 2 minutesFor 35 cycles:98°C for 30 seconds for denaturation30 seconds for primers annealing at temperature depending on primers and polymerase72°C for 30 seconds per kb of DNA for elongationFinal extension:72°C for 7 minutes4°C on holdFor 35 cycles:98°C for 30 seconds for denaturation30 seconds for primers annealing at temperature depending on primers and polymerase72°C for 30 seconds per kb of DNA for elongationFinal extension:72°C for 7 minutes4°C on holdFinal extension:72°C for 7 minutes4°C on hold
?. [Gene specific PCR]
Prepare the master mix using the quantities specified in Materials
?. [Gene specific PCR]
Control the PCR by doing an agarose gel electrophoresis (follow Gel Electrophoresis Protocol)
?. [Gene specific PCR]
Load the PCR mixture in thermocycler and start program.
?. [Extension PCR]
Prepare the master mix with the gene specific PCR product and using the quantities specified in Materials
?. [Extension PCR]
Load the reaction mixture in the thermocycler and start the program
?. [Final PCR]
Control the PCR by doing an agarose gel electrophoresis (follow Gel Electrophoresis Protocol)
?. [Final PCR]
Purifiy the PCR product with a DNA purification kit
?. [Final PCR]
When the extension PCR is done, directly add in the tubes the primers for the final PCR with the quantities specified in Materials.Minimize the waiting time before starting the second reaction to keep polymerase activity high