BIMM 100 Lecture Notes - Lecture 8: Sanger Sequencing, Primer Walking, Restriction Enzyme
31 Jan 2018
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Dna replication in a cell vs. in a test tube. In a cell: rna primer synthesized by primase. In gel electrophoresis, when dna fragments are small, easy to separate because speeds are very different. However, when dna fragments are large, difficult to separate because speeds through gel are very similar. Why you can only sequence ~1000bp: methods to use sanger sequencing, primer walking: add primers every ~1000bp so you can sequence each section individually. Disadvantage is that it takes a long time: shot gun: overlap dna and sequence each section at the same time. Increase temperature to 72 c to allow elongation of primers. How to design primers: design one primer that anneals to bottom strand 5" -> 3", desig(cid:374) a(cid:374)othe(cid:396) p(cid:396)i(cid:373)e(cid:396) (cid:272)o(cid:373)ple(cid:373)e(cid:374)ta(cid:396)y to top st(cid:396)a(cid:374)d that goes f(cid:396)o(cid:373) 5" -> 3". If you want to ligate pcr product into vector, can use restriction enzymes: cut vector with restriction enzymes. Incorporate restriction site sequence to beginning of primer sequence.
