Mini Prep
what is binding affinity?
The strength of the interaction between two molecules.
Only ____ fragments can be ran on a gel
Linearized
Why is Taq polymerase efficient to use for PCR reactions?
Thernostability
What are the two types of ways restriction enzymes can cut
Explain why over‑mixing the lysis before neutralization is not encouraged
Over‑mixing can shear genomic DNA
Do smaller fragments of DNA migrate exponentially faster or slower on a gel?
Due to their ability to navigate more easily through the pores of the agarose gel.
Why is the denaturation step necessary in each cycle of PCR?
To create single-stranded DNA for primer binding.
Why would you purify a PCR product before running a gel?
To confirm the PCR amplification was successful
Why do we perform a dry spin ?
To remove ethanol from wash buffer & any other contaminants
On what end do I load my DNA sample?
Cathode end (black)
How do you know the annealing temperature to set the thermocycler at ?
Tm-5
How are plasmids linearized for cloning and analysis ?
enzymes!
Explain how elution buffer works
UNBINDS DNA (low salt concentration)
Make a 3% gel using 175mls of 1X TAE solution
5.25g
Name two real world applications PCR is used in
COVID testing & Forensic analysis etc.
Can I clone on an area that is not MCS of a vector?
Yes, some vectors do not contain MCS & just encode for restrictive enzymes.
Why does DNA bind to the GeneJet Column?
under high salt concentration, DNA binds w/sillica membrane
What would I see if I ran an undigested plasmid on a gel?
Multiple bands. Undigested plasmids can exist in multiple confirmations
Name ways you can troubleshoot your PCR
-primer design, thermocycler concentrations, DMSO etc.
Why might some plasmid have multiple selectable markers?
Experiments with multiple plasmids, increases efficiency and accuracy. etc.