PCR and Cloning Flashcards
Why plateau effect in PCR
Thermodynamic phenomenon - large proportion of amplicon increases melting temp
Steps in PCR
Initial denaturation (activates fast Taq)
Denaturing (95-98 - formation of single stranded DNA)
Annealing (temp decrease = primer bind, need correct Ta)
Elongation (72 - DNA polymerase extend primers to produce nascent DNA, 30s/bp)
Final extension (72, 5mins)
Hold (4 degrees)
Essential components of PCR (8)
Template DNA Sequence specific primers (fwd and rev) dTNPs Reaction buffer 1X MgCl2 (enzyme cofactor) ddH2O DNA polymerase
Wallace rule
Tm = 4(G+C) + 2(A+T)
What can influence Tm of primers
Monovalent cations
Divalent cations
Flanking nucleotides
Salt concentration
How to create 3’ stability
Avoid 3’ mismatch
3’ base should be a G or a C
(Avoid more than 3 G/Cs at 3’, forms unwanted complexes and could give false priming)
Two types of 2dry primer structures
Intraprimer - self-complementarity (hairpin)
Interprimer - primer dimers
What to do if PCR doesn’t work
Ta gradient
Mg2+ titration (Mg cofactor for Taq, BUT binds DNA affecting primer template interaction and Taq primertemp interaction) there’re more Mg = less stringent bonding
Limitations of PCR
Need sequence and flanking info
High risk of contam
Can only amplify a certain length of DNA (5kb is easy, up to 40kb)
Three strategies for cloning PCR products
- Blunt end PCR cloning - proof reading DNA poly (pfu) generates blunt ends
- T/A cloning - non-proof reading DNA poly(Taq) have terminal transferase activity (adds adenine on end of product)
- Restriction site end cloning - NB add bases to 5’ end so RE can bind.
Fx of poly histidine tags
Allow protein purification by affinity chromatography
Allow semi-quantitative analysis by immunoblotting
Choice of vector depends on
Nature of insert
Downstream applications
Expression vectors contain
Promotor
Selection marker
N/C terminal His tags or fusion proteins
Ori
Describe reporter gene vectors
Gene of protein easily quantitated (eg. GFP, luciferase) linked to regulatory sequence being tested (eg. Promotor or fusion protein) Changes in transcriptional activity detected by changes in level of reporter gene expression
What is special about shuttle vectors
They can replicate in more than one organism (appropriate oris)
Define directional cloning
Cutting both vector and insert with two different REs. Ensures insert is inserted in one direction. Efficiency greatly improved.
How do you convert a 5’ overhang to a blunt end
Use Klenow and T4 DNA polymerase in presence of dNTPs to fill overhang.
How do you convert a 3’ overhang to blunt end
Use exonuclease activity of T4 DNA polymerase in presence of dNTPs
What is a linker
A small dsDNA fragment that contains a restriction site. Can convert blunt to sticky.
NB ensure restriction site is not at end of linker
What does DNA ligase do
Forms new phosphodiester bond between 5’ P and 3’ OH. Requires energy.
What does Alkaline Phosphatase do
Removes 5’ phosphates from vectors preventing self ligation. Reduces non-recombinants
Why NB to calculate vector insert ratios
Only two ends to any DNA despite varying size. Therefore one must take into account length when working out number of available ends.
How to calculate molar ratios
Look at pink paper
What are satellite colonies
Tiny colonies appearing close to true Abx resistant colonies. Due to reduced Abx around true clone.
How does blue/white screening work
E. coli plasmid contains lacZ which codes for B-galactosidase. Active B-gal hydrolyses lactose to glucose and galactose. lacZ gene contains multiple cloning site, therefore when insert present = no functional protein. Agar contains x-gal, if x-gal cleaved by B-gal than colony turns blue.
