Optimization Of PCR Flashcards
When may a hot start be need for PCR
When a thermophile polymerase like Taq isn’t used, need to activate enzyme and start at 95 degrees
Name the basic things to optimise to optimise your PCR reaction
Optimal [Mg2+]
Correctly calculated annealing temp.
Correct extension time (short frag. = short time)
Primer (re-)design - banding, primer dimer etc.
Additives e.g. BSA or Betanine
Explain significance of annealing temp.
Provide correct temperature for enzyme to bind specific strands and NTs
Have to be calculated (Tm)
Must be confirmed practically
Sometimes compromises between yield and specificity
Explain the significance of [Mg2+] on PCR
The fidelity of PCR depends on it. Cofactor for DNA polymerase that stabilises dsDNA and raises Tm. Important for controlling specificity.
Describe characteristics of gel electrophoresis
Workhorse method after PCR
Looks at amplified fragments for contamination
Separated through matrix
Relies DNAs neg. Charge to move to positive terminal when current is out through.
Separates based on size like chromatography
Loading buffer (bromophenol blue) used to help see and the glycerol weights sample to bottom of well.
When troubleshooting PCR, how can you correct incorrect product size
Primer design may be off so there’s mis-priming
- too low annealing temp
- Redundant sequences
When troubleshooting PCR, how can you correct no PCR product
No priming
- too high annealing temp
- primer dimers (primers annealing to primers [complementary sequences])
- too much template
- forgot enzyme/dNTPs
- incorrect [Mg2+]
What does BSA do as a PCR additive
(cheapest) Stabilises Taq and overcomes PCR inhibitors
What does Gkycerol do as a PCR additive
Increases apparent concentration of primer/template mix and often increases PCR efficiency at higher temps
What do stringent enhancers do as additives to PCR
Enhance yield, reduce non-specific priming
What do Non-ionic detergents do as additives to PCR
Stabilise Taq and suppresses formation of secondary structure
Describe the process of real time PCR
Quantitative PCR. PCR cDNA flourescently labelled primers.
SYBR-Green 1 binds to dsDNA and the complex emits green light (522nm)
Fluorescence increases proportionally as DNA is amplified (expression)
Signal is given off when reaction reaches exponential phase.
Quicker it reaches e phase, the more gene is being expressed
Describe the feature of real time PCR
Quantitative PCR.
Can monitor expression of certain genes over time in res. To treatments.
Multiplex - monitor multiple at same time
Signal can be quantified
Shows gene expression
How can real time PCR be used to determine optimal [MgCl2]
Do the same reactionjn different salt conditions and conclude best condition by which one reaches e phase quicker
Name some variations of PCR
Transcriptase PXR
hot start PCR
Nested PCR
Touchdown PCR
