mol7 - BIMM 100 - continuing Chapter 5 (except 5.4) !...

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BIMM 100 -- continuing Chapter 5 (except 5.4) Midterm Exam Thursday 29 April, in class Location: stay tuned Check with your TA for Week 5 OH/Sections Today: PCR hybridization in all its glory making good stuff Thursday: inactivating genes ** Steve Gilmore We will not cover 5.4 before the Midterm Review Session - Tues night Help Rooms Mon & Wed afternoons Check WebCT for schedule & materials Additional office hour this week Wednesday 2-3pm in 2130 Pacific Hall Practice Midterm will be posted Thursday on WebCT
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vectors (plasmids) restriction endonucleases, DNA ligases poly-linkers libraries (oligos) cloning fnding a clone/gene defning (describing/sequencing) a clone PCR learning From hybridization: blots, in situs , arrays . ...... expression cloning
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Sequencing: Action! http://www.dnalc.org/resources/animations/cycseq.html http://www.wiley.com/college/pratt/0471393878/ student/animations/dna_sequencing/index.html
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DNA sequencing -- getting more information Imagine a λ phage clone, 25 kb insert primers (vector seq.) region sequenced for more sequence …. new primers new sequence ʻ walking ʼ -- successively building sequence on basis of new information vs. ʻ shotgun ʼ sequencing -- a collection of overlapping small fragments( subclones ) ʻ assemble ʼ sequence computationally When an insert is ‘too big’ for a single sequencing run Two different strategic approaches
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The polymerase chain reaction (PCR) Selective amplifcation oF DNA sequences Relies on knowledge oF DNA sequence and in vitro DNA polymerization template (target) DNA synthetic oligonucleotide primers with homology to ends oF targeted sequence dNTPs thermostable DNA polymerase means oF controlling the reaction(s) Needed Length ~ 20bp (4 20 ~ 10 12) Base composition G:C stability Perfect vs near matches cloning, forensics, identification
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The Polymerase chain reaction (PCR) Denature template with primers and polymerase Primers anneal (base pair) Polymerase copies lower temperature MCB 5-23
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PCR uses cloning -- Figure 5-24 probe preparation -- forensics diagnostics -- Classic experiments template (target) DNA oligonucleotide primers dNTPs + * dNTP thermostable DNA polymerase means of controlling the reaction(s) * * * * * * * * * * * Limitations error-rate
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This note was uploaded on 06/03/2010 for the course BIMM Pillus taught by Professor Molecularbiology during the Spring '10 term at UCSD.

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mol7 - BIMM 100 - continuing Chapter 5 (except 5.4) !...

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