Lecture9mcb160Lf11

Lecture9mcb160Lf11 - Really Serious PCR problems and...

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Really Serious PCR problems and artifacts: Trouble shooting 1. Nothing works i.e. No product Check reagents using positive control ; change reagents/enzyme if necessary Is your template DNA clean? Use new template if necessary. Change parameters for Thermocycler: Reduce annealing temperature for primers, or increase the number of cycles; try different primers. 3. Everything works the same way i.e. Same product all the time Even negative control (without DNA template) gives the same product. Reason: Contamination , usually by previous PCR product or by plasmid DNA 2. Non-speciFc bands Use higher annealing temperature, try new longer primers.
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3. (cont’d) Contamination problems - What to do Throw out all reagents and start all over with fresh reagents, template Change pipettors (could be aerosol problems) and any other equipment that could be contaminated In extreme cases, UV irradiation of reagents to break the contaminating molecules How to avoid contamination- Take precautions: Keep your reagents and template DNA samples away from any ampliFed PCR products Prepare small working stocks of reagents, and discard periodically Use separate pipettors, tubes, bench for setting up PCR reactions Use plugged pipet tips
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The D1S80 locus: located on chromosome 1, function unknown Contains VNTR (Variable Number Tandem Repeat) The repeating sequence at D1S80 is 16 bp in length (see p. 85 of Lab Manual) At least 29 different alleles known Range from 14X to 45X repeats ( covering ~220 bp to ~700 bp); 23 repeats most common HHMI, Princeton University VNTR genotyping of human DNA by PCR 143 bp 64 bp PCR Applications: DNA fingerprinting
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Each of us has two copies of the D1S80 locus, one inherited from each of our parents. ~86% of the population is heterozygous at D1S80 because a different VNTR allele has been inherited from each parent. HHMI, Princeton University
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Amplified D1S80 PCR products: Homozygous individual A has one band and heterozygous individual B has two bands www.Edvotek.com A B
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Summary of Subcloning Experiments Two Subcloning experiments 1) directional cloning using an insert cut with two different restriction enzymes and
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Lecture9mcb160Lf11 - Really Serious PCR problems and...

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