User:Noah Benjamin/Notebook/Experimental Biological Chem/2011/09/20
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The base pair sequences of the forward and reverse primers was solved (using the Quick Change Manual Website) such that the melting temperature of the primer did not fall below 78 degrees. Using sterile lab practices, 43µL of double distilled water, 1µL of a 10mM DNTP, 1µL of a 50 ng/µL template DNA, and 1.25µL of a 100ng/µL primer mix, 1µL of enzymes, and lastly 25µL of a wax compound were added together. These were placed in the thermocycler and run as follows: 1 cycle of 95˚C for 30 seconds 16 cycles of 95˚C for 30 seconds, then 55˚C for 1 minute, then 68˚C for 3.6 minutes (3 minutes 36 seconds). This was left to cool at 4˚C overnight.
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