amplified DNA sequence of 800 bp promoter UGA -3. For this we use the PCR technique , components to accomplish this are:
Buffer dNTP
MgCl 2 First
DNA pol ( Taq polymerase , thermostable , survive high temperatures)
mold genomic DNA
H2O concentrations calculated to ensure that all of this in 150 ul
* A mix when everything is important to note that the first thing you should put the water and the B uffer because other components are very sensitive to place in the vacuum tube and nothing happens to them.
prepare three tubes with 50 ul PCR each mixture. Two of them with the mold (we will call positive (+)) and one without it (let's call this negative (-)). Tube is necessary to negative because it indicates whether there was contamination or not and whether what I have in the tubes is only gene DNA gel UGA also -3 or more. We want to ensure only be amplifying the selected sequence.
To make PCR is a program where everything is heated to 94 ° C (to separate DNA chains ) lowering the temperature to 63 ° C (at this temperature are joined primers) and then rises to 72 ° C (the temperature optimum for polymerization ). This program is done in cycles amplify and have a lot of product.
Buffer dNTP
MgCl 2 First
DNA pol ( Taq polymerase , thermostable , survive high temperatures)
mold genomic DNA
H2O concentrations calculated to ensure that all of this in 150 ul
* A mix when everything is important to note that the first thing you should put the water and the B uffer because other components are very sensitive to place in the vacuum tube and nothing happens to them.
prepare three tubes with 50 ul PCR each mixture. Two of them with the mold (we will call positive (+)) and one without it (let's call this negative (-)). Tube is necessary to negative because it indicates whether there was contamination or not and whether what I have in the tubes is only gene DNA gel UGA also -3 or more. We want to ensure only be amplifying the selected sequence.
To make PCR is a program where everything is heated to 94 ° C (to separate DNA chains ) lowering the temperature to 63 ° C (at this temperature are joined primers) and then rises to 72 ° C (the temperature optimum for polymerization ). This program is done in cycles amplify and have a lot of product.
Program used: 94 ° C 5
min 94 ° C 1 min \\
63 ° C 1 min ) is repeated 30 times.
72 ° C 2 min /
72 ° C 5 min
min 94 ° C 1 min \\
63 ° C 1 min ) is repeated 30 times.
72 ° C 2 min /
72 ° C 5 min
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