molbio lab set-up Flashcards
3 areas in the spatial separation of pre and post amplification of work areas
- sample preparation r
- reagent preparation r
- amplification r
air pressure in each areas
reagent prep - positive
sample prep - negative
post-amplification - negative
single entrance, reagent used for amplification should not be exposed to other areas.
what area is this specifically?
reagent prep
alternative to spatial separation
- class II biological safety cabinet
- dedicated areas for each work phase
- unidirectional
- automated specimen processing station
other lab design to be considered
- temperature and humidity requirements
- exhaust ventilation
- water quality
- electric outlet
- back-up power system
- eyewash
- ergonomic assessment
laboratory practices
- use positive displacement pipettes and disposable filtered pipette tips
- avoid production of aerosols when pipetting
- use of sterilized single use plasticware
- use of cleanroom sticky floor mats
- minimizes the risk of amplicon carry-over on clothing, hair, and skin
- clean punches between samples
- use of nuclease-free or autoclaved water
- aliquot oligonucleotides
- always include a blank control to check contamination
- use of electronic data system
- wipe test (swab test)
this test is to detect, localize, and remove contamination. to identify the source of the contamination. this is done monthly
wipe test (swab test)
cleaning materials for PCR station
- UV light
- 70% ethanol
- 10% sodium hypochlorite
- DNA away
substitution of uracil for thymine during PCR amplification
enzymatic inactivation with uracil-N-glycosylase
cleaning of chemical and enzymatic controls
- work stations should be cleaned with 10% sodium hypochlorite
- ultra-violet light irradiation
- enzymatic inactivation with uracil-N-glycosylase
false amplification potential causes
- non-optimized assay conditions
- unknown polymorphisms in target sites
- oligonucleotide concentrations too high
- nucleic acid cross-contamination
in labeling reagents, it should have __
- lot no.
- expiration sate
- storage requirement
- date of use/disposal
enumerate the use of molbio lab
- health
- medicine
- forensics
- pharmaceutical industry
- biological warfare
- drug discovery
- PCR based technology
- fluorescence in situ hybridization (FISH)
- biochip
- peptide nucleic acid (PNA)
- proteomic technology
- electrochemical detection of DNA
contamination may cause
- incorrect results
- require extensive cleanup
- loss of creditability
- impact on financial and performance
enumerate the importance of accurate pipetting
- quantitative precision
- reproducibility
- data quality
- resource efficiency
false amplification potential causes
- non-optimized assay conditions
- unknown polymorphisms in target sites
- oligonucleotide concentrations too high
- nucleic acid cross contamination
types of pippets in a molbio lab
- micropipette
- multichannel pipettes
- serological pipettes
sizes of micropipettes that we used
P10 (0.5-10uL)
P20 (2-20uL)
P100 (10-100uL)
P1000 (100-1000 uL)
micropipettes are essential for?
- PCR
- DNA quantification
- sample transfers
these pipets allow for simultaneous pipetting of multiple sample, they are commonly used in high-throughput applications
multichannel pipets
these pipets are used for transferring larger volumes, typically in milliliter range.
serological p
serological p are often used for?
- media preparation
- cell culture
- larger-scale molbio experiments
enumerate the pipetting techniques
- calibration
- pipette selection
- pre-rinse
- aspiration and dispensation
- tip ejection
- avoid air bubbles
- changing tips
- vertical pipetting
- touch-off technique
- sample mixing
proper way to avoid cross-contamination when pipetting multiple samples
always change tips between samples
which is more accurate? forward or reverse?
forward
it is the preferred method when exact volumes are critical such as qPCR or DNA quantification
forward p
reverse pipetting is used for?
working with viscous or volatile fluids
principles of forward pippeting
- accurate
- preventing contamination
principles of reverse p
- minimizing viscosity and volatility effects
- reduced error
this technique reduces the risk of over-pipetting and ensures that the intended volume is accurately delivered
reverse p
pipet techniques used for qPCR, spectrophotometry, DNA quantification. also suitable for general sample transfers and dilutions
forward pipetting
this technique is ideal for transferring concentrated glycerol stocks or volatile solutions which can evaporate during pipetting
reverse
often used in the preparation for DNA/RNA extractions
pH measurement in molbio is essential to maintain?
accuracy and reproducibility
neutral pH:
acidic pH:
alkaline pH:
neutral: 7
acidic: <7
alkaline: >7
accurate pH measurement is used for?
- buffer preparation
- enzymatic reactions
- cell culture maintenance
components of a pH meter
- electrode
- meter/analyzer
- buffer solutions
- reference electrode
core component of the pH meter which contains a glass membrane sensitive to pH changes
electrode
pH values of buffer solutions
- 4.01
- 7.00
- 10.01
buffer solutions of ph meter is used for?
calibration
proper usage of a ph meter includes (8)
- calibration
- electrode conditioning
- sample preparation
- rinse between measurements
- temperature correction
- electrode maintenance
- avoid contamination
- interference
when to perform calibration?
before each series of measurements or when using a diff electrode
this helps to stabilize the electrode and maintain its performance
electrode conditioning
what sol’n to use when rinsing the electrode?
deionized water
why is it important to maintain proper records of pH measurements?
to ensure data integrity and traceability
measure of the concentration of a solute in a solution
molarity
molecular weight of a substance expressed in grams
moles
1 molar = ?
1 mol/L = 1M
___ measures the amount or quantity of material; ___ measures the concentration of that material in a solution
moles measures the amount or quantity of material; molarity measures the concentration of that material in a solution
Avogrado’s number
6.022x1026
weight of the solute as a percentage of the volume
weight/volume
formula in calculating dilutions
concentration stock x volume of stock uses = concentration final x final volume
product of PCR
amplicons
for cheap lab that cannot afford spatial separation?
separation of pre-PCR to post-PCR
purpose of spatial room
to avoid contamination
in spectrometry, ____ means contaminated
presence of bands
enumerate the lab equipments for molbio that we have in the laboratory
- gel electrophoresis
- PCR/thermal cycler
- microcentrifuge
- micropipette/serological pipet
- pH meter
- dry heat blocks
- class II biosafety cabinet
- refrigerator
used to separate mixtures of DNA, RNA, or proteins according to molecular size
gel electrophoresis
used to amplify target nucleic acid sequences into millions of copies
PCR
used to heat or cool the samples in a controlled manner
dry heat blocks
convert:
2000 mg = __ g
what is the conversion factor?
2g
conversion f: 1000
convert 5L to __ mL
5000 mL
convert 198 ug to __ pg
198 000 000 pg
convert 500 mM to ___ M
0.5 M
convert 75 uL to __L
0.000075 L
convert 65g to __ mg
65 000 mg
convert 0.9 nM to ___ uM
0.0009 uM
convert 5.6 g to ___ ug
state the conversion factor
5 600 000 ug
cf: 1 000 000
20 mM = ___uM
20 000 uM
convert 100 uL to __ MI
0.1
to make a 2% agarose gel (w/v), how much agarose would you put into 50 mL of 1X TAE buffer?
formula: vol of sol’n x density of sol’n
solution: 50 mL x 1g/mL
= 1 g
for: total mass of sol’n x desired %
sol: 50 g x 0.02
= 1 g
serves as a clean bench area
dead airbox with UV light
proficiency testing is performed __
twice a year
enumerate the potential sources of contamination
- cross-contamination between specimens
- amplification product contamination
- laboratory surfaces
- ventilation ducts
- reagents/supplies
- hair, skin, saliva, and cloths
