01:119:117 Lecture Notes - Lecture 7: Cytidine, Thermal Cycler, Tae Buffer
7 May 2018
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Midterm Exam Review
Lab #1/ Lecture #1
• Sterile technique (aseptic technique) = a way a to keep all your experiments
free from contaminants.
• Labeling= section #-Group # Initials-description of sample
o Example: 26-1SA-P
o Labeling petri dish = ALWAYS LABEL THE BOTTOM BECAUSE THE
LIDS CAN BE SWITCHED
• Assay= a tool used in science that helps quantitatively measure the amount of
a single part of a total sample
o Example: a glucometer determines the amount of glucose in the blood
• Data must be exact and reproducible!
• Accuracy= how close a measurement is to its true value
o Correctness
o The closer the arrow is to a bulls eye the more accurate the shot is
• Precision= the ability to repeatedly measure a value in a fixed situation and
get the same results
o Reproducibility
o If multiple shots are fired at a bullseye, precision would be the cluster
of arrows
• Significant figures= digits that carry meaning contributing to its
measurement resolution
o Keeps track of quality of measurements
o Important to use sig figs when recording a measurement so that it
does not appear to be more accurate that the equipment is a capable
of determining
o If the # before the 5 is odd, round up.
o If the # before the 5 is even, round down
o None of your data or calculations should have more decimal places
than what the instrument measures (should never exceed 3 decimal
places)
• Metric System Units
o 1ml =1000 ul
o Meter= 1/10 millionth the distance from the pole to the equator
o Units of volume and mass were derived from the meter
o Metric unit of mass, the gram, is defined as the mass of 1 ml of water
▪ Mega=one million=10^6
▪ Kilo=one thousand=10^3
▪ Milli=one-thousandth=10^-3
▪ Micro=one-millionth=10^-6
▪ Nano=one-billionth=10^-9
▪ Pico=one-trillionth=10^-12
o Examples:
▪ .18 ml -> 180 ul (multiply by 1000)
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▪ 150 mg -> .15 g (divide by 1000)
▪ if you are given 2.5 ul of a 10pmole/ul solution of DNA how
much pmol do you have? 2500000
• Pipet Bulbs= measure 1-10 milliliters
o After you use them they are no longer sterile
▪ Do not let them touch the surface
▪ Most contamination occurs by not being prepared
o Squeeze the air valve (marked A) and with other hand squeeze large
bulb to expel air
o Slowly draw up liquid by releasing the A button and squeezing the S
button
o Press E to expel the liquid to wear you want to release the liquid
• Transfer Pipets=transfers liquids that are less than 1 ml
• Pipetman (measure in microliters)
o P-20= 1-20ul
▪ 4.2 =042
▪ 5.8=058
▪ 173=17.3
o P-200=20-200ul
▪ 137=137
▪ 039=39
▪ 178=178
o P-1000=200-1000ul
▪ 093=930
▪ 046=460
▪ 109=1090
• Percent Error (%)=theoretical mass-measured mass/ theoretical mass *100
o If greater than 2% pipetting was not that accurate
• Making buffers and dilutions
o Buffers= an aqueous solution containing a mixture of salts, buffering
agents, and sometimes reducing agents, detergents, or cofactors, in
which each of the components has a purpose and is included to
optimize the reaction
▪ function to resist changes in hydrogen ion concentration
▪ basic function of a buffer is to resist chnages in hydrogen ion
concentration
o Stock solutions= concentrated solutions that last over long periods of
time
▪ Take up less space
▪ Easily diluted
• The following equation can be used to make a specific volume of a dilute
solution from a stock solution: C1V1=C2V2
o C1= concentration of stock solution
o V1= volume of stock solution needed to make dilute solution
o C2= final concentration of dilute solution
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o V2= final volume of dilute solution
▪ Dilution Factor- factor by which the concentration of the dilute
solution is reduced compared to the concentration of the stock
solution.
▪ Example: Prepare 100 ml of 10mM Tris buffer from a 1M Tris
stock and determine the dilution factors
• Rearrange C1V1=C2V2 to solve for the unknown
o V1=C2V2/C1
▪ V1=unkown
▪ C1=1M
▪ V2=100ml
▪ C2=10mM
▪ V1=(10mM)(10ml)/(1000mM)
• V1=1 ml
o Determine volume of water needed
▪ Vwater=V2-V1
▪ Vwater=100ml-1ml=99ml
o Calculate the dilution factor
▪ DF=V2/V1= (100ml)/(1ml)
▪ DF=100
o A dilution factors of 100 means that the 10mM
Tris buffer has a concentration 100 times, or
100-fold, lower than the 1M tris stock.
▪ Solutions labeled with X
• Indicates that the solution is in a concentrated form that
must usually be diluted to a 1X concentration for use.
o Example: 5X concentrated must be diluted 5-
fold, while a 100 concentrated solution must be
diluted 100-fold
• Example: Prepare 1 liter of 1x TBE buffer from a 10X
TBE stock solution
o V1=
o C1=10X
o V2=1liter
o C2=1X
▪ Calculate V1=(1X)(1liter)/(10X)
• V1=.1 liter=100ml
▪ Calculate Vwater=V2-V1
• Vwater=1000ml-100ml=900ml
o Therefore, to prepare 1 liter of 1x TBE from a
10X TBE stock solution, you should add 100ml of
10X TBE stock to 900ml of water
• Serial Dilution
o A stepwise dilution where the stock solution for each dilution in the
series is the dilute solution from the previous dilution
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