Lecture 7 - Lecture7...

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Lecture 7 PCR - when you anneal primers in PCR you must know the T m T m --> the measure of the stability of the structure, whether it is RNA-RNA, DNA- RNA, DNA-DNA. Dependent on the length the of the hybrid and the GC content of the hybrid To ensure that the generated hybrid is created with the sequence that is desired, the general rule is to set the Annealing temperature to 3 degrees less than the T m for the your primers or template Each cycle result in double the amount you started with (2 n ) Sanger Dideoxy DNA sequencing Manual DNA sequencing, each ddNTP reaction is run on a separate lane. Se- quence is read bottom to top. each reaction generates all potential fragments that terminate at a given base. Idea is that if your template is homogenous you can never have two different nucleotides in the same position, you can only have one nucleotide in a given position. So that the fragment lengths between lanes cannot be the same be- cause it would mean that the dideoxy terminated at the same position for two different nucleotides, which is not possible. original primer decides where you start sequencing smallest fragment, is the fragment closes to the primer. actually reading the sequence of the complementary to the template strand Automated Big Dye terminator DNA sequencing difference between this and manual DNA sequencing has to do with the gel and fragment length dideoxy has a fluorescent dye that is distinguishable by a laser. Instead of running a slab gel, it runs all four reactions after they are done separ- ately. You mix the reactions together and run a capillary gel so that all the reac- tions are on the same length, then the capillary has a collection tube to elute the bands of the capillary gels as they are running off. As they run it, so there is only one fragment in one drop, then the laser shines light on the drop and you can see fluorescent tag did that fragment had. Then get a chromatogram. Migration goes in fragment length
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Lecture 7 - Lecture7...

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