Protocol 3 - Restriction Digest

in this example DNA samples in which the oxt our locus has been amplified will be digested with restriction enzymes to determine genotype first the amplified samples are needed including the negative control the appropriate restriction enzyme is also needed in this case the variant being studied in the oxt our gene demonstrates alternative digestion by bam h1 enzymes must stay on ice at all times or they may lose their cutting activity obtain as many new tubes as you have DNA samples as always mix the amplified samples and collect the solution in the centrifuge make sure your new tubes are labeled and transferred 10 microliters of amplified solution into the corresponding tube amplified samples can be put in the fridge at this point do not throw them away they will be needed as the comparison and control during gel electrophoresis add one microliter of the restriction enzyme to each tube be aware of the small volume but also the viscosity of the enzyme solution enzymes solutions typically contain glycerol a very thick solution it will stick to the tip which could cause the addition of more than as needed into the reaction wipe the tip on the edge of the tube before dispensing into your sample repeat this process for the remaining samples cap the tubes and label them to distinguish from the amplified samples mix and centrifuge to collect the solution place the samples in the thermocycler if necessary which for oxt are it is run the cut program remembering to enable the heated lid and watch for errors until the cycler has reached temperature

Channel: The Jackson Laboratory

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Keywords: dna, sequencing, genetics, genomics

Id: sEjN_fxJN1s

Length: 5min 49sec (349 seconds)

Published: Thu Jul 02 2015