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Exam 1 Flashcards

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1
Q

What are the three parts of Alkaline-SDS Preps?

A

Solution 1: Resuspension buffer

Solution 2: Denaturation Buffer

Solution 3: Neutralization buffer

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2
Q

Alkaline-SDS Preps: Resuspension Buffer

A

Tris - pH (alkaline)

EDTA - chelates divalent cations (Mg2+, Ca2+) - inhibits DNAses

Glucose - maintain osmotic pressure - prevents lysis

RNAse A - digest RNA

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3
Q

Alkaline-SDS Preps: Denaturation Buffer

A

NaOH - breakdown cell wall, disrupt H-bonds of DNA (denature - ssDNA), disrupts protein

SDS - solubilize cell membrane and forms white precipitate with cell debris and denatured protein

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4
Q

What are the major concerns of Alkaline-SDS Preps?

A

cross-contamination from another source, sheering of genomic or large plasmids

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5
Q

Alkaline-SDS Preps: Neutralization Buffer

A

Potassium acetate & Glacial Acetic Acid - decrease pH & restore H-bonds in DNA (renaturation - dsDNA)

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6
Q

Phenol Extraction

A

Aqueous portion - contains nucleic acids - DNA/RNA

Phenol portion - contains protein

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7
Q

What are the main concerns of Phenol Extraction?

A

cross-contamination from another source, sheering of genomic or large plasmids - RNA degradation by RNAses

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8
Q

Cesium - Chloride Preps

A

Density gradient - very pure preps and large quantities

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9
Q

What are the major concerns of Cesium - Chloride Preps?

A

cross-contamination from another source, sheering of genomic or large plasmids - RNA degradation by RNAses

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10
Q

What are the ways of DNA and RNA concentration?

A

Ethanol Precipitation

SpeedVac - Vacuum Concentrator

Columns (chromatography) - Anion-exchange columns, Oligo dT cellulose, Sephadex and sepharose

Beads

Carriers

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11
Q

What are the types of ethanol precipitation?

A

Sodium acetate

Sodium Chloride

Lithium Chloride

Ammonium Acetate

Salt

Ethanol

Temperature

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12
Q

DNA/RNA Concentration: Ethanol Precipitation: Sodium Acetate

A

0.3M final conc, pH 5.2

for routine DNA precipitations

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13
Q

DNA/RNA Concentration: Ethanol Precipitation: Sodium Chloride

A

0.2M final concentration

for DNA samples containing SDS since NaCl keeps SDS soluble in 70% ethanol so it won’t precipitate with the DNA

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14
Q

DNA/RNA Concentration: Ethanol Precipitation: Lithium Chloride

A

0.8M final concentration

for RNA

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15
Q

Why is Lithium Chloride used in ethanol precipitation for RNA?

A

This is because 2.5-3 volumes of ethanol should be used for RNA precipitation and LiCl is more soluble in ethanol than NaAc so will not precipitate, but beware - chloride ions will inhibit protein synthesis and DNA polymerase so LiCl is no good for RNA preps for in vitro translation or reverse transcription. In these cases use NaAc

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16
Q

DNA/RNA Concentration: Ethanol Precipitation: Ammonium Acetate

A

2M final concentration

for the removal dNTPs, but do not use for preparation of DNA for T4 polynucleotide kinase reactions as ammonium ions inhibit the enzyme

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17
Q

DNA/RNA Concentration: Ethanol Precipitation: Salt

A

neutralizes negative charge on phosphate backbone - helps precipitate out of the water

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18
Q

DNA/RNA Concentration: Ethanol Precipitation: Ethanol

A

Decreases the dielectric charge compared to pure water - helps the salt to precipitate the nucleic acid

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19
Q

DNA/RNA Concentration: Ethanol Precipitation: Temperature

A

cold temperature increases flocculation (colloids come out in suspension)

  • overnight @ -20C
  • 30 min @ -70C (70% ethanol/dry ice bath)
  • 5 min on dry ice
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20
Q

DNA/RNA Concentration: SpeedVac - Vacuum Concentrator

A

centrifuge that runs under vacuum with a heater - evaporation , so decreases the water content of the sample

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21
Q

DNA/RNA Concentration: Anion-exchange Columns

A

positively charged diethylaminoethyl cellulose (DEAE) groups on the resin’s surface interacts with phosphate backbone

separates by charge

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22
Q

DNA/RNA Concentration: Column Chromatography: Oligo dT cellulose

A

purification of mRNA (poly-A tail)

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23
Q

DNA/RNA Concentration: Columns Chromatography: Gel Filtration

A

Separates by size

Sephadex and Sepharose Columns

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24
Q

DNA/RNA Concentration: Beads

A

Magnetic polymer or streptavidin beads that bind the nucleic acid - theory of isolation is similar to columns - bind nucleic acid, was, elute

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25
DNA/RNA Concentration: Carriers
Sometimes carriers are added when nucleic acid concentration is very low → nucleic acid (e.g. tRNA) or glycogen are common
26
What is the absorbance (260/280 nm) of dsDNA?
1A260 = 50 ug
27
What is the absorbance (260/280 nm) of ssRNA?
1A260 = 40 ug
28
What is the absorbance (260/280 nm) of DNA?
1.8
29
What is the absorbance (260/280 nm) of RNA?
2.0
30
What are the steps of PCR?
Template denature → Primer Anneal → DNA synthesis → repeat cycle
31
Steady - State PCR
take a reading after every cycle
32
RT-PCR
33
nested PCR
amplify one set, design primer during → use as a template
34
Semi-Nested PCR
Design one new primer and use with one old primer
35
Southern Blot
Looking for Genes in genome use primers and sequence to locate where in genome, gene is
36
What are the steps of Southern Blot?
digest with restriction enzyme →Run RNA/DNA → electrophoresis → transfer nucleic acids to filer with salt solution → use radioactivity (hybridized with unique 32p - labeled nucleic acid probe) →remove unbound probe → expose X-ray film to filter
37
What happens if you don't digest genomic DNA with restriction enzyme before you run a southern blot?
it will smear
38
Northern Blot
Used for looking at expression of RNA load markers/RNA/DNA → electrophoresis → migration → transfer nucleic acids to filter with salt solution → use radioactivity (hybridized with unique 32p - labeled nucleic acid probe) →remove unbound probe → expose X-ray film to filter
39
Western Blot
Load separation gel → blot → incubate with antibody → develop autoradiogram
40
DNA Sequencing
use ddNTPs→ put one ddNTP in each tube → run → use bands to get sequence
41
Modern DNA Sequencing
each ddNTP is given a color → run through laser → order of colors corresponds to sequence
42
Gene Expression: Plasmid Map
Promoter for gene → 5' primer site → restriction site → inserted gene → restriction site → 3' primer site → origin of replication → antibiotic resistance gene → selectable marker
43
Gene Expression: Plasmid Preparation
see image
44
What are the ways to transduce a cell?
heat shock - bacteria transfection - tissue culture cells infection - bone marrow cells
45
Gene Expression: Transduction
anyway you get foreign DNA into a cell
46
Gene Expression: Transduction: Transfection
calcium - phosphate precipitation - form Ca and P crystals that help DNA move across the membrane Cationic Lipid - Lipid helps get across membrane Electroporation - current helps cross membrane
47
Gene Expression: Transduction: Infection
retrovirus/lentivirus
48
Protein Isolation - Cell Lysis
Physical Detergent Protease inhibitors
49
Protein Isolation - Cell Lysis: Physical
homogenization - grind up detergent - anionic, cationic, zwitterionic, and non-ionic Protease inhibitors - proteins are very susceptible to degradation
50
What are the major concerns of Protein Isolation - Cell Lysis?
degradation by proteases, alteration of protein modifications
51
Protein Quantification
BCA, Lowry, Bradford Assays - colorimetric Absorption at 280 nm - aromatic rings on amino acids Biuret - peptide bonds
52
Immunoprecipitation
shows protein - protein interaction pull down protein A → western blot with protein B → if protein is there A&B interact
53
what are the steps of immunoprecipitaiton?
suitable antibody is added → antibody binds to protein of interest → Protein A or G added to make antibody-protein complexes insoluble → Centrifugation of solution pellets antibody-protein complex → removal of supernatant and washing → western blot
54
Aseptic Technique
a way of working that maintains sterility → critical for tissue culture work
55
When is sterile technique required?
living organisms or with buffers, media, culture containers for living organisms
56
When is sterile technique recommended?
Molecular biology techniques
57
air currents
carry dust, spores, and pathogens
58
arms/hands
bacteria, yeast, etc
59
What is the best practice for aseptic technique?
• Avoid drafts and traffic • Do not crowd working area • Wipe down before and after use (70% ethanol – made with 190-proof) • All supplies need to be sterile • Minimize hand movement/motion (creates air current) • Wear gloves – sterilize often; when in doubt – change the gloves • Minimize pouring – creates aerosols and fluid on the lip of the bottle • Open bottles at ~45-degree angle • Removing cap – place FACE-UP • Flaming – for bench-top only – quickly flame glass pipettes, bottles, etc (use of a flame in the tissue culture hood is discouraged) • Do not leave bottles open • Work with one cell line at a time
60
What are the “Don't Dos”?
• Pipette too far into the filter • Touch the tip of pipette to anything (bench top, tissue culture hood, bottle, etc) – “you bump-it, you replace it” • Reuse pipettes (reusable glass pipettes must be cleaned and plugged before they can be reused)
61
What are the types of cell cultures?
primary cells continuous cell lines transformed cell lines hydbridoma adherent cells suspension cells
62
Cell Cultures: Primary Cells
isolated from animal tissue
63
Cell Cultures: Continuous Cell lines
immortalized
64
Cell Cultures: Transformed cell lines
tumorigenic (either isolated from tumor tissue or cell lines have been transformed in the lab)
65
Cell Cultures: Hybridoma
fusion of antibody-secreting B cells and malignant myeloma cells
66
Cell Cultures: Adherent Cells
grow in monolayer, attached to the surface of the tissue culture vessel
67
Cell Cultures: Suspension Cells
grow suspended in the tissue culture medium
68
Observing Cell Cultures: Color of medium
yellow - acidic, bacterial contamination or cell overgrowth magenta - alkaline, fungal infection or CO2 issue
69
Observing Cell Cultures: Cloudiness/smell
indicates contamination usually yeast
70
Observing Cell Cultures: Cells
peeling cells - overgrown adherent cells clumping - overgrown suspension cells
71
Observing Cell Cultures: cross-contamination
are there multiple cell types in the same plate?
72
Where are Frozen Cell Stocks often stored?
liquid nitrogen long-term
73
What is in the storage buffer for frozen cell stocks?
medium with increased serum concentration and DMSO
74
what does DMSO do for frozen cell stocks?
prevents the formation of ice crystals
75
How are frozen cell stocks used?
• Cells are pelleted by centrifugation, media removed and pellets resuspended in freeze-down solution
76
How are frozen cell stocks maintained?
* Freezing must occur slowly (1 – 3°C per minute in order to minimize cell damage) * Thaw quickly (warm water bath) – immediately dilute freeze- down media with cells, pellet and resuspend cells in fresh media
77
Type II Biosafety Cabinet
78
What are the steps for working sterilely?
* Verify the hood is on and air is circulating – 20 min before use if not kept on 24 hours or if no UV light * Work at the sash level * Do not block airflow!!! * Secure light-weight materials * Wipe-down work area and sash of the hood before and after each use * Do NOT use a flame in the biosafety cabinet
79
Mycoplasma
\*worst contamination you can have - Small; can’t be seen using standard inverted microscope - No cell wall – not harmed by most antibiotics - Usually notice because viability decreases and cell performance starts to change - Should routinely screen cell lines – fluorescent staining, PCR, send to a company
80
What cells are generally the first effected by mycoplasma?
Primary cells
81
How do you count cells using a hemacytometer?
add 10 uL of cells → Count the 4 corners (16 squares each corner) - take the average of cells = average x 104
82
What is the surface area for growth for suspension cells?
the media to calculate: dilution factor of final volume → if dilution factor is 1:5 and final vol is 30 → take 1/5 of 30 = amount of old culture needed
83
What is the surface area for growth of adherent cells?
plate media is only food
84
What is the process to dilute adherent cells?
1. remove media 2. trypsonize (detach cells from plate) 3. stop enzyme reaction → add media (at least double amount of trypsin added) → volume you dilute from **_volume you dilute from_** vol = amt trypsin + amt of media (needs to be at least double trypsin) ex: vol = 1 mL trypsin + 4 mL media = 5 mL total (this is what you dilute from) to dilute: dilution factor (as fraction) of new vol if you want to dilute 1:5, final vol of 10 mL 1/5 of 5 mL = 1 mL cells + 9 mL media

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