Quiz 2 PPTs - contd Flashcards by Gail I

PCR lab:
cycling steps:
1. denature: … - … degrees C, … DNA strands

90; 95;

separates

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PCR lab:
cycling steps:
2. anneal: … - … degrees C, …

45; 65;

primers anneal

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PCR lab:
cycling steps
3. elongate: … degrees C, at this stage … to the …

72;

polymerase adds complementary nucleotides; growing chain

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PCR lab:

Denaturing also … and is needed for …

improves polymerase;

activation

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PCR lab:

annealing temperature depends on … of …

melting temps;

primers

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PCR lab:
annealing temp:
simplest form = …
primers (i think) should be within … degrees of each other

4(G + C) + 2(A+T)

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PCR lab:
annealing temp:
- too low: …
- too high: …

nonspecific;

nothing anneals

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PCR lab:
elongation:
time depends on … (…)

length of amplicon; copied DNA

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specific temps needed for actin:

denature: … deg. C
anneal: … deg. C
elongate: … deg C

95;
52;
72

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uses for PCR: 
... (this is why we're doing it)
... 
... 
.../... 
...

cloning expressed sequence; 
cloning; 
genotyping; 
expression; presence; 
mutagenesis

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TOPO-TA cloning: 
using E. coli: 
- isolated from ... 
- rod shaped
- ... chromosome, < ... bp, ... genes

intestines;
circular;
5 million;
4000

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TOPO-TA cloning: 
reasons to use E coli: 
... 
... 
... 
... amounts

simplicity;
quick generation time;
convenience;
large

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TOPO-TA cloning: 
reasons to use E coli: 
simplicity: 
- ... genes 
- ... 
- ...

4000;
single cell;
identical

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TOPO-TA cloning:
reasons to use E coli:
quick generation time: … (mammalian cells …)

20 mins;

1 day +

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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

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TOPO-TA cloning: 
what to use E coli for:
- ... - make the bacteria make copies of the DNA for us, ... 
- ... 
- ...

cloning; proofreading;
libraries;
bioremediation

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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