Experiment 2  Time is based on:

o Efficiency of Taq polymerase

1. 3 microfuge tubes: into each, place 6l restriction buffer o Length of sequence
 Detergent- disrupt nuclear and cell membrane  so you can extract
Tube No. 1 2 3 DNA
Restriction buffer 6 6 6  Protease- degrades histones and nuclease
 phage DNA 4 4 4  Salt- disrupt histone-DNA complex without it ,lower yield
Pst 1 1 0 0  Ethanol- needed to precipitate DNA;
EcoR 1 0 1 0
Hind III 0 0 1 Source organism Cleaves what
Eco Rq E. coli G * AATTC
2.  phage DNA: 4l of 0.2g/l into 3 separate tubes Hind III Haemophillus influenzae A * AGCT
3. Pulse spin the tubes Pst I Providencia stuartii CTGCA * G
4. Incubate at 37C for 30 minutes  store in freezer -20C until next
meeting Sample loading dye
5. Pulse spin reagents before use; keep reagents in cool temperate (ice  If dye is lighter, DNA will be behind and dye will go ahead
box w blue ice) Components
 Glycerol- make your sample dense so when you load it in the well,
Retrovirus- RNA virus sample will sink and not float in the buffer
 It’s okay to ingest because it doesn’t come w the enzymes that would  Tracking dye (Eg: Xylene cyanol, bromophenol blue, orange G) there
allow it to insert itself into your DNA can be 1 or more of these in your dye; so samples wont overrun the
gel
Restriction enzyme
 Cuts the DNA at specific sites at specific sequences (restriction site)  Oral report- max 20 minutes
 Restriction sites usually 4-6 bases long
o …GGATCC…  …GG ATCC… Electrophoresis
…CCTAGG…  …CCTA GG…  Separate DNA fragments according to size and charge
 Sticky ends- middle  Light DNA will travel faster
 Blunt ends- outside  Affects DNA movement
 Search how they name restriction enzymes o Voltage
o Gel concentration
Gel: o Buffer strength: 1X (less ions so slower migration)
 1X TAE- prepare 1L from 50X stock  Higher buffer strength  faster migration
o Tris o DNA conformation
o Acetic acid  Linear
o EDTA  Circular
o TBE = tris + boric acid + EDTA  Nicked- circular w putol
o Temperature
C1V1 = C2V2  You can load 6-7l per well
1(1000mL) = 50(X)
20mL stock of 50X = X Experiment 3
20mL + 980 mL dH2O Bradford Assay- formal report
1. Dilute milk samples 50-fold
 100X Fast Blast stain instead of ethidium bromide 4l sample
o 200mL from 500X stock 196l PBS phosphate buffered saline
1(4) =
100(200) = 500(X) Chuckie = 4.98 g protein
40 = X 2. 20l diluted sample + 1000l Bradford reagent
40mL + 160mL dH2O 3. Standards

Store at room temperature [protein] in g/mol Volstd VolBR1000 Abs595

Expt: mg/mL mL
1. Prepare & load agarose gel 0.125 20 0.097
a. 1% agarose gel 0.25 20 0.174
i. 0.5g agarose
0.5 20 0.432
ii. 50mL 1x TAE
0.75 20 0.514
iii. Heat but don’t allow to boil, just until clear; then
cool 1.0 20 0.595
b. Black: anode and (-) 1.5 20 0.675
c. Red: cathode and (+) 2.0 20 1.296
d. pH 8 yung TAE buffer (-) so it’ll travel (-) to (+) so your C1- chuckie 0.679
wells should be at the (-) C2- chuckie 0.665
e. About 15 minutes to polymerize; don’t move it (opaque C3- selecta 0.361
means it’s polymerized) C4- selecta 0.351
2. Prepare samples (from the freezer)
a. To each tube, add 2l of sample loading dye  pulse spin BR1000 = Bradford
b. 6 buffer, 4 DNA, 1 restriction enzyme, Std = standard = sample
3. Run gel, 100V until tracking dye runs 2/3 of gel (30-45 mins) Incubate for 5  60 minutes
a. You can increase voltage to speed it up, but it will heat 5 minutes is ideal; too long and results aren’t nice
the sample and it can degrade your samples (remedy: put Read the absorbance at 595 nm/A595
it in the ref to lower the temperature)
4. Staining  Plot [protein] and absorbance
a. Add 100X Fast blast enough to submerge the gel for 2  X-axis: [protein] (it's known)
mins. w shaking (gently)  Y-axis: absorbance
b. Wash gel w water (40-55 C) for 10 seconds (destaining  Consider dilution factor (50-fold) so multiply result by 50
the gel)  Standard is not within the range of the graph
c. Time in this step is crucial o Lower left of graph  lower dilution
d. Wash gel with tap water (every 5 minutes) until bands in o Upper right of graph  increase dilution
the gel become visible (destaining the gel) o Then repeat the experiment because incubation time
e. Only the DNA is stained by Fast blast should be the same
 Milli q water- very pure  Standard curve is only valid within its range
o No nuclease- degrade DNA  Outlier:
o No protease- degrade Taq  Micropipette:
 o No lower limit, assume min is 20% of the max b. Reaction mix: 40 L + 10 L Orange G
3. 10 wells; we can use up all the wells (made of polystyrene)
Abstract 1 a. Each has 10 L
Intro 2 b. Determine which of the unknowns pertains to a certain
MM 2 antigen
Results 3 4. ELISA
Discussion 4 a. 10 ml 1X PBS
Conclusion 5 b. Assay
Recomm 3
Postlab
Experiment 4  Mean is easily affected by extremeties, so median is used as a better
Polymerase Chain Reaction (PCR) measure of central tendencies
 Amplification of certain DNA fragments  Lyophilized- concentrated
 Some uses: crime scene investigation
 You will be given DNA from 4 suspects Experiments
 Job: amplify certain regions of the DNA of the suspects and then  Basic instruments and techniques
determine who the guilty one is  Virtual microarray
 Solution:  Bradford assay
o DNA- template  Size exclusion chromatography
o Master mix (MM):  ELISA
 MgCl2- cofactor of Taq (Taq requires it to  DNA isolation
function properly  RFLP
 dNTPs- for growing chain  PCR and AGE
 Buffer- To maintain optimum conditions to for
PCR to occur Basic instrument
 Primers- start 1. Bio Ref
 Taq polymerase- Catalyze the polymerization a. Ultra ref freezer- -80
reaction (extension); has inherent deficiency 2. UV/Vis spectrophotometer- absorbance
a. To determine: DNA concentration and purity (is it DNA
only or is it contaminated?)
Methodology b. Extract DNA  dissolve DNA is proper buffer
1. Prepare DNA template 3. Photo documentation system- photo of gel
a. 20 L each template DNA into PCR tube 4. Micropipettor (0.5-10microl, 2-10microl, 20-100, 100-1000 microl)-
2. Prepare MMP (master mix w primers) transfer and measure
a. 125L MM + 2.5L primer 5. Thermal cycler
3. Reaction mix 6. Gel electrophoresis system
a. 20L DNA + 20L MMP a. Horizontal gel electrophoresis
b. After preparation  freezer; make sure tubes are properly b. Vertical gel electrophoresis
labeled 7. Shaker/agitator- for cell cultures to agitate continuously (nutrient and
 Always prepare template and MM separately cultures are mixed)
 Always prepare more MM than you need 8. Vortex mixer- mix
 In excess: dNTPs 9. Microcentrifuge- separate components acc. to density
 Correct amount: primers, Taq (expensive) 10. Water bath
11. Distilling apparatus
PCR 12. Autoclave/sterilizer
1. Denature- break strand of DNA template at 94C for 30s 13. Ice shaver
2. Adjust temp- DNA template + primer anneal 14. Parafilm
a. Annealing temp. depends on the primer sequence and
length DNA quantification and purity assessment via spectrophotometry
b. 52C for 30s A260 = 1 for:
3. Adjust temp- Taq polymerase can extend 50g/ml dsDNA
a. 72C for 1 minute 33g/ml ssDNA
4. Repeat 35 times (in experiment) 40 g/ml RNA
 Before beginning:
o Initial denaturation step: 94C for 2 minutes to ensure that A260/A280 :
all your 2 stranded DNA molecules are separated Get ratio of absorbance at 260 and 280
 At the end: 2- pure RNA
o Final extension: 72C for 10 minutes 1.8- pure DNA
o To ensure that all PCR products are fully extended 0.6- pure protein
 Emergency and you have to leave: Ideal for DNA: 1.8-2.0
o Hold (pause) 4C for infinity (you can leave it as long as
1 𝐴260
you need to) =
 Repeat for 30-40 cycles (normally) 50 𝑥
 Machine will adjust the temperature, but 1st versions made use of
water bath Sample solution: plug in: A260 = 0.361  find X
X = 0.361 x 50 = 18.05
 In the freezer: -20C
[DNA] = x (df)
Exercise 6
df = total volume/spl vol
Size Exclusion Chromatography (SEC)
Example:
Protein mix contains:
 Quartz Cuvette (not plastic): 50l sample + 950 buffer;
Hemoglobin (Hb) Brown 65kDa
 Example if you got: A260 = 0.361
Vit B12 Pink 1350Da
 X = 18.05 g/ml
Exclusion Limit (of the beads) = 60kDa  “lulusot lang siya through the beads”  [DNA] = 18.05 (20) = 361 g/ml
ELISA (Enzyme-linked immunosorbent assay) o X x DF 
1. Agarose gel
a. 0.5g agarose Conversion of units of the centrifuge
b. 50 ml 1 X TAE N = [(1.118 x 105 x g)/r]1/2
2. Samples G = RCF = 0.00001118rN2
a. LD: Orange G  RPM- changes with the size of the rotor
i. Stock: 5X  In a journal, use g so that others can follow the method
ii. Needed: 1X  M1 V1 = M2 V2
Where:
  N = rotating speed, rpm o Even if not target region, there is specific binding between
 g = rel. centrifugal force template and primer
 R = rotational radius, cm o Primer was bound somewhere it shouldn’t have
 Only low MW bands appear
Bradford Assay o Around 20 base pairs
 To determine the protein concentration in milk samples o Since other primers formed primer dimers  none
amplified
Principle  No bands appear
 Protein determination method that involves the binding of Coomassie o Improperly stained
Brilliant Blue G-250 dye to proteins o Expired stain
 Dye exists in 3 forms: o Wash step: water temp. was diff.
o Cationic- red o TAQ was not added
o Neutral- green o Poor primer design (wrong primer sequence)
o Anionic- blue
 Acidic conditions: predominantly in doubly protonated red cationic
form (Amax = 470 nm)
 Upon binding to protein, converted to a stable unprotonated blue
form (Amax = 595 nm)
 CBBG dye binds primarily to basic (esp Arg) and aromatic amino
acid residues  explains residues

Size Exclusion Chromatography

 Beads in column have tiny pores
 The mixture of molecules is added to the column
 Large molecules move through the column quickly travelling around
the beads
 Smaller molecules move through the pores of the beads and take
longer to pass through the column
 Column bed- mass of beads in the column
 Beads trap orsieve and filter molecules based on size
 Fractionation- separation of molecules

PHOO

ELISA
12 well strip made of polystyrene

PBS- phosphate buffer saline

Gentle tube inversion- avoid bubbles

RFLP
 Tubes, incubate
 Something was used as a marker because the sizes are known;
when run in gel, you can determine the sizes of the fragments that
resulted from the digestion
 Uses: barcoding
 Alelle 1: get 3 fragments
 Allele 2: get 2 fragments
 Semi-log: get log of size; measure distances travelled by the
fragments  use the curve to determine sizes of fragments from the
digestion
 What happens if primers are palindromic?
o These will bind  primer dimer (bind instead of
elongating sequence)

DNA isolation: Phenochloroform ?  very pure dna

STR- used to differentiate a person from another

STR analysis- fastest and efficient

Real life:
 Consider 3 loci, but all of them are STR  you can better
differentiate 1 person from another
o Get the power of discrimination
 Allele frequencies:
o Different in different races: Caucasian, Asian, etc

Our PCR: 1 primer + master mix

Possible change: Duplex, multiplex (Addition of more than 1 primer)
 Primers need similar annealing temp., and shouldn’t be
complimentary (or else will form primer dimer)

Other possible scenarios (where your experiment may have gone wrong:
 Lower number of bands than expected
o Annealing temp. may have been too high
 More bands than expected
o Possible contamination
o Annealing temp. may have been too low