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24 Cards in this Set
- Front
- Back
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Why does a hot start increase product yield? |
It limits polymerase activity to reduce non-specific amplification. |
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What is the problem with adding the enzyme at lower temperatures? |
Non specific products can be formed. |
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What is a PCR specific example of a positive control, negative control, and blank. |
Blank: Rnase free water Positive: Expected yield from primer manufacture Negative: Omitting template DNA |
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What effect does multiplex PCR have over basic? |
It allows 2+ DNA targets to be amplified. |
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When RT-PCR takes place what is the product lacking compared to other types of PCR? |
Introns |
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Why is primer size important? |
If it is too short can bind to multiple sites, if too long slows down reaction. |
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What is 1 positive and 1 negative to using Pfu over Taq? |
It has incorporated proofreading to to correct errors. It is prohibitively expensive. |
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What 2 functions do Magnesium ions perform in a PCR reaction? What must be avoided when using Mg and why? |
Stabilizes Primer/template interaction Stabilizes polymerase Avoid EDTA forms compunds with Mg |
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What is the relationship between the number of cycles in the thermocycler and the amount of DNA produced? |
More cycles=more DNA |
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What is a hot start? |
When all components of PCR are added except the enzyme then heated to annealing temperature, then adding the enzyme. |
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What are the 3 types of cell transfer and what medium do they use? |
Transfection - introduction to mammilian host transduction - virus DNA transfer Transformation - introduction via bacterial cell |
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What is a competent cell? |
A cell that can take in DNA |
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Describe electroporation |
high voltage electro pulses create pores in cell wall, high concentration of plasmid outside goes into the cell. |
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Describe a gene gun |
tungsten or gold bullets coated with dna shot into a cell |
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Describe Microinjection, with drawbacks and positives. |
Microcapillary needle inserted directly into cell. - difficult and time consuming +optimum DNA can be injected |
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Describe what a restriction enzyme is and the 2 types of ends. |
Restriction enzymes recognize specific seqeuences and cleave the phospor diester bond. Blunt straight ends, sticky ends |
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What is the difference between blunt and sticky ends? |
Blunt are straight and general, sticky seperates certain base pairs allowing new sequences to be attached. |
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How do bacteria stop their DNA from being digested? |
They methylate bases in the recognition sequence. |
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Explain epigenitics |
Determines how genetic code is expressed by methylating certain areas. |
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What is required for PCR? |
Template, dNTP, primers, DNA polymerase, Mg buffer |
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Explain the 3 steps of thermocycling and the duration of each. |
Denaturation melts DNA into 2 single strands at 94C Annealing allows primers to attach, temp is cooler than melting temp of DNA Extension Allows polymerase to extend the primer each interval takes 1 minute |
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What happens if there are to many dNTP's? |
Error rate increases and the taq polymerase may be inhibited |
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What happens is the primer is too long or too short? |
if too long it will slow the reaction, if too short can bind to multiple non specific site |
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What is a transgenic and knockout animal |
transgenic = extra gene knockout =missing |
