Quiz 2 PPTs - contd Flashcards
PCR lab:
cycling steps:
1. denature: … - … degrees C, … DNA strands
90; 95;
separates
PCR lab:
cycling steps:
2. anneal: … - … degrees C, …
45; 65;
primers anneal
PCR lab:
cycling steps
3. elongate: … degrees C, at this stage … to the …
72;
polymerase adds complementary nucleotides; growing chain
PCR lab:
Denaturing also … and is needed for …
improves polymerase;
activation
PCR lab:
annealing temperature depends on … of …
melting temps;
primers
PCR lab:
annealing temp:
simplest form = …
primers (i think) should be within … degrees of each other
4(G + C) + 2(A+T)
PCR lab:
annealing temp:
- too low: …
- too high: …
nonspecific;
nothing anneals
PCR lab:
elongation:
time depends on … (…)
length of amplicon; copied DNA
specific temps needed for actin:
denature: … deg. C
anneal: … deg. C
elongate: … deg C
95;
52;
72
uses for PCR: ... (this is why we're doing it) ... ... .../... ...
cloning expressed sequence; cloning; genotyping; expression; presence; mutagenesis
TOPO-TA cloning: using E. coli: - isolated from ... - rod shaped - ... chromosome, < ... bp, ... genes
intestines;
circular;
5 million;
4000
TOPO-TA cloning: reasons to use E coli: ... ... ... ... amounts
simplicity;
quick generation time;
convenience;
large
TOPO-TA cloning: reasons to use E coli: simplicity: - ... genes - ... - ...
4000;
single cell;
identical
TOPO-TA cloning:
reasons to use E coli:
quick generation time: … (mammalian cells …)
20 mins;
1 day +
TOPO-TA cloning:
reasons to use E coli:
large amounts: 10^9 cells/ml
convenience: easy to … in lab, minimal … and … required, can be stored at … for weeks
maintain;
media;
temp;
4 deg. C
TOPO-TA cloning: what to use E coli for: - ... - make the bacteria make copies of the DNA for us, ... - ... - ...
cloning; proofreading;
libraries;
bioremediation
TOPO-TA cloning:
what to use E coli for:
-production of …
recombinant proteins
TOPO-TA cloning:
what to use E coli for:
- production of recombinant proteins - lots of protein, low cost
however, if produce too much too quickly:
- … proteins - in …, little to no …
misfolded;
inclusion bodies;
activity
TOPO-TA cloning: what to use E coli for: - production of recombinant proteins. however, if produce too much too quickly: - misfolded proteins - ... - no ...
toxicity;
post translation modification
TOPO-TA cloning:
how we use E coli:
- … bacteria: needed to create … bacteria strains with … probability of surviving outside of host
disabled;
safe;
low probability
TOPO-TA cloning:
how we use E coli:
- disabled bacteria - early strain did not … or …
- now strains are made for …
grow well;
take up plasmid;
specific purposes
TOPO-TA cloning:
how we use E coli:
- now strains are made for specific purposes: highly efficient for …, propagating … and …, high …, or expression of …
taking up DNA;
cDNA; genomic libraries;
copy numbers;
recombinant proteins
TOPO-TA cloning:
transformation:
- process by which genes are transferred into a cell as …
- for our lab, transformation is the introduction of … into …
free molecules of DNA;
plasmid DNA;
bacterial cells
TOPO-TA cloning:
transformation:
- for us - introduction of plasmid DNA into bacterial cells (not same transformation as normal cell to cancer cell, or transfection, or DNA to animal or plant cell)
- DNA + .,.. bacteria, which means that the bacteria can …
competent;
take up DNA
TOPO-TA cloning:
transformation:
2 types of competence for bacteria: … or …
chemically competent;
electrocompetent cells
TOPO-TA cloning:
competent cells:
- chemically and electro differ in how they… and how they …
were made;
take up DNA
TOPO-TA cloning: competent cells: chemically competent: - ... colony, grow ..., 30 deg C or lower - then ... the bacteria
single;
slowly;
harvest
TOPO-TA cloning: competent cells: chemically competent: - harvest cells - ... in high concentration of ... and ... (this ... and ...)
resuspend; Ca2+; other salts;
damages cell wall;
makes holes
TOPO-TA cloning: competent cells: chemically competent - after resuspending, ... and freeze - this method is less ... but also has ... and takes ...
aliquot;
expensive;
lower efficiency;
30 min longer
TOPO-TA cloning: competent cells: electrocompetent: - also single colony, grow slowly, 30 degrees C or lower - then harvest - no ...! ... - aliquot and freeze
salt;
glycerol
TOPO-TA cloning:
competent cells:
electrocompetent:
- to introduce DNA into cells use …, which will then … and allow DNA in. This process, though …
electricity;
open holes in the cell walls;
kills half of the cells
TOPO-TA cloning: competent cells: electrocompetent: - ... efficiency - more ..., sensitive to ...
higher;
expensive;
salt
TOPO-TA cloning: antibiotic resistance: how we select for the bacteria that have taken up the plasmid: - ... - ..., ... , ... - ...
ampicillin;
chloroamphenicol; streptomycin; kanamycin;
tetracycline
TOPO-TA cloning:
antibiotic resistance:
- ampicillin: reacts with enzymes that … No cell wall –> no …
build cell wall;
bacterium
TOPO-TA cloning:
antibiotic resistance:
- chloroamphenicol, streptomycin, kanamycin - bind …, …
bacterial ribosome;
inhibit translation
TOPO-TA cloning:
antibiotic resistance:
- tetracycline - also binds …, but resistance is conveyed by expression of a …, … out of cell
ribosome;
sugar transporter;
pumps tet.
TOPO-TA cloning:
antibiotic resistance:
- ampicillin reacts with …, which helps with building the cell wall. leads to …
transpeptidase;
cell lysis
TOPO-TA cloning:
antibiotic resistance:
- ampicillin resistance = expression of …, which cleaves … (which is what’s in ampicillin), inactivating the antibiotic
beta-lactamase;
4 member ring
For TA cloning, from PCR product choose:
- …
- …
single band;
correct size
What other DNA could be in the messed up PCR samples:
- …
- …
- …
pcr product;
primers;
nucleotides
bc pcr products were messed up, what to do for TOPO TA?
- Run +RT samples out on gel, …, …
- magically have PCR product appear
cut out band;
gel purify dna
the correct bacterial colonies to use from topo ta are the …, … colonies
well isolated; not blue
