Feb 27 Flashcards
(23 cards)
What are the steps and temperatures for PCR?
Have denaturation at 94, then annealing at 50-65 ish, then elongation at 72. This cycle will repeat may times
How many duplexes are there present from 1 dsDNA strand after 5 rounds of PCR, how many are the same size?
There are 32 duplexes (64 individual strands but 32 dsDNA strands). 22 duplexes are the same size.
Why is DMSO added to PCR mixtures?
It lowers the melting temperature of DNA, disrupts H-bonding
Can plasmid or genomic DNA be in lower amounts for PCR start?
You need more genomic DNA than plasmid templates for PCR to be successful
What are primer-dimers? How prevent this?
It is when primers have micro-homology, so they can pair with other primers, or form hairpins. As they are small, they amplify very efficiently. Always happens, so need to use correct combination of primers to prevent this.
What is the equation to roughly determine primer melting temp Tm?
This is when 50% of primers are perflectly duplexed, 50% are not. It is 4 degrees C for each G and C, and 2 degrees for each A and T. So primer that is GAGTAG is 3 G’s, 3A/T, so (4x3)+(2x3) = 18 degrees ?
What does Mg2+ do for PCR? What happens if too low or high?
It reduces the electrostatic repulsion of phosphate backbones, so stabilizes the annealing of primers to target. Too high or low causes more non-specific binding. More Mg2+ will speed up polymerization as more stable but less accurate.
What happens if primer binds non-specifically in PCR?
It will be elongated like usual to match the incorrect template strand it bound to. Then for the next round of PCR this new strand formed with the primer will match another primer, but has incorrect template as well. This gets amplified so lots of product could be this
What is 3’ complementarity in primer design?
It is when is is better that the primer complements the template at the 3’ end than the 5’ end. So if 5’ end of primer not bound to DNA, it is sort of ok, but if 3’ end does not complement template, it won’t work
On what end of primer strands to you add restriction enzyme sites? What else add to RE site sequence?
Because 3’ complementary base pairing is more important, add RE sites to 5’ end of primer. RE sites can’t be at very end as it is endonuclease, so need to add some bases so the RE sequence starts a few from the 5’ end.
What is the ideal length, GC content, Tm of primers?
Ideally 18-30 nucleotides, could be longer for more specificity. Want 40-60% GC content, poor binding if too low, false binding if too high. Want Tm to be 55-70 degrees, primers should have similar Tm.
Primers should have ___1___ in the last 5 nucleotides (____2____)
Primers should not have any __3__ with each other or pair with ___4___.
1= 1-3 G/C’s
2=GC clamp
3= complementarity
4= Itself
What polymerase used for PCR, why?
Taq polymerase, it is from bacteria in Yellowstone hot springs, optimal at 70 degrees.
Does Taq pol work at 94 degrees when strands are annealed? How fast is it at adding bases (processivity), what is it’s fidelity?
It is stable but not active at 94 degrees, best around 70 degrees. It is fast at adding bases, but low fidelity as no 3’-5’ exonuclase activity so no proofreading
Why are all reagents for PCR kept on ice until PCR is run? Why add the ______ last?
Primers and templates will non-specifically pair an Taq pol will polymerize at room temp, so keep reagents on ice until last minute and add polymerase last.
What is Hot-start PCR?
It is when Taq pol is bound to antibody, so first round of PCR is longer. But once antibodies are degraded, then activated once hot and primers have annealed properly
For cloning and long sequences of PCR, do we still use Taq? If not what?
No, it does not have proofreading. So use other high temp polymerases with proofreading ability, bit slower but at least more accurate. Have Pfu and Phusion.
What is 2-step PCR?
If annealing temp is above 72 degrees, then polymerization and annealing happens at same time, so 2 steps not 3 steps in the cycle
What is gradient PCR?
Each tube will have different temperature, so can see which will be best for highest yield but keeping # cycles same as more cycles also give more inaccurate replications
How restirction enzyme site is made so can be inserted into plasmid?
PE sequence and primer do PCR like usual. Have blunt ends, so then do RE digest, will cut a bit on the strand so now have sticky ends for ligation to plasmid
What is colony PCR?
It is when mix colony with PCR miz, can screen colonies for presence of insert or something on gel
What is genotyping?
It is when examine single gene for mutations. Do reverse transcription PCR, qPCR and sequencing
