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Knight:Annealing and primer extension with Klenow polymerase: Difference between revisions
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Knight:Annealing and primer extension with Klenow polymerase (view source)
Revision as of 08:01, 11 May 2006
, 11 May 2006→Procedure
Barry Canton (talk | contribs) |
Barry Canton (talk | contribs) |
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#*Y μL oligo 2 (typically 1 μg or more) | #*Y μL oligo 2 (typically 1 μg or more) | ||
#*(87 - X - Y) μL deionized sterile H<sub>2</sub>O | #*(87 - X - Y) μL deionized sterile H<sub>2</sub>O | ||
#Anneal the two oligos together by either placing the mixture in a thermal cycler ([http://www.mjr.com MJ Research], PTC-200) at 94°C for 5 mins, a cool down for 0.1°C/sec to | #Anneal the two oligos together by either placing the mixture in a thermal cycler ([http://www.mjr.com MJ Research], PTC-200) at 94°C for 5 mins, a cool down for 0.1°C/sec to 5°C below the melting temperature of the primers, hold that temperature for 5 mins, then cool down at 0.1°C/sec to 37°C. Alternatively, the tube can be placed in a beaker of boiling water and let cool to room temperature. | ||
#Add 1 μL Klenow 3'<math>\rightarrow</math>5' exo<sup>-</sup> polymerase to mixture. <br> Vortex polymerase before pipetting to ensure it is well-mixed. | #Add 1 μL Klenow 3'<math>\rightarrow</math>5' exo<sup>-</sup> polymerase to mixture. <br> Vortex polymerase before pipetting to ensure it is well-mixed. | ||
#Add 1 μL dNTPS (equal to 0.25 mM final concentration of each dNTP). <br> ''Recommend using a thermal cycler for the following incubation steps.'' | #Add 1 μL dNTPS (equal to 0.25 mM final concentration of each dNTP). <br> ''Recommend using a thermal cycler for the following incubation steps.'' | ||
