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PRIMER DESIGN
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Developed by Kary Mullis
Few DNA strands to millions of copies
Repeated heated and cooling
What is polymerase?What is chain reaction?
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'My God, you could use
this to isolate a fragment
of DNA from a complex
piece of DNA, from its
context.
KARY MULLIS
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Making a PCR reaction
•Taq DNA polymerase •dNTP mixture•PCR buffer (salts, loading dye, etc)•MgCl2•ddH2O•DNA template•Primers
http://www1.qiagen.com/Products/Pcr/TaqSystem/TaqPcrCore.aspx
http://www-che.syr.edu/faculty/boddy_group/pages/thermocycler.jpg
http://upload.wikimedia.org/wikipedia/commons/8/81/PCR_tubes.png
Primer Design
PRIMER LENGTHOptimal length – 18 – 22 bp
MELTING TEMP52 – 58 CGC content determines temp
NO SELF COMPLEMENTINGFormation of hairloop structuresIf 3’ ends complement, form primer dimers
AVOID C OR G RUNSHarder to separate
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Sample QPCR Readout
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LIGASE CHAIN REACTION
BACTERIAL
CLONING
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LACKS SELF - CORRECTION
HIGH SENSITIVITY
SHORT DNA
SEQUENCES
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