Lab 5/6 Flashcards
(i.e., restriction digest, )
which creates double-stranded cuts in DNA using restriction enzymes
- Know how to calculate Accuracy and Precision for a pipetman
Accuracy= Experimental mean/true value * 100
Precision= Standard deviation/Experimental mean * 100
- Know how to determine the expected # of fragments and the expected average size of those
fragments from a restriction digest
Calculate number of sequences present by multiplying probability of each nucleotide together and dividing number of total nucleotides by the odds of getting that sequence
- Know how to calculate how much of each component to add to a restriction digest (enzyme,
template, water, buffer)
C1V1=C2V2. 10x to 1x- 10 is referring to relative concentration. (10X) (v) = (1x) (1)
V=.1
- Know how to calculate the required amounts of agarose or buffer for a certain percentage of
agarose gel
% agarose * total volume (mL) /100
- Know the basic procedural details for a restriction digest and making a gel
- Set up gel casting system
- Melt agarose in hot buffer
- Add DNA stain and swirl flask
- Let gel cool
- Pour gel into gel casting tray, add comb, let solidify
- Remove comb and gel, use or store with buffer.
- Know how to calculate how much loading dye to use for a given volume of sample to load on a gel
C1V1=C2V2
- Know the general procedural details for a ligation and transformation
- Be able to interpret a gel image of undigested and digested human genomic DNA and plasmid DNA
- Why are we running a gel??
To determine whether the digest was successful- separating fragments by size lets us ________
- Why do we want to run uncut DNA on the gel?
- Why ampicillin?
Kills off cells that weren’t transformed
- Why X-gal?
It’ll show which E coli cells were recombined (Ones that weren’t transformed will be blue because of X-gal)
- Why IPTG
Induces LacZ expression
agarose gel electrophoresis,
separates DNA fragments based on their size
loading dye
the sample heavy so it sinks into small indentations, called wells, within the gel and also provides a color to visually track the sample as the gel is running
cloning vector,
which will carry the DNA into the host organism
selectable markers.
Selectable markers aid in identifying bacterial cells that have taken up a plasmid and which of those are recombinant plasmids
recombinant plasmids
(plasmids carrying a foreign DNA insert)
ligase.
Ligase will seal the pieces of DNA together through the creation of phosphodiester bonds between the 5’ phosphate of one nucleotide and the 3′ OH of an adjacent nucleotide
transformation,
which is a process in which the host organism, E. coli, takes up the plasmid.
β-galactosidase
breaks down a substrate called X-gal, which yields a blue color.
restriction
endonuclease/enzyme
recognize specific nucleotide sequences and make double-stranded cuts within those sequences
