Lab1 : DNA isolation

1. Detergent lyses the membrane while

1. Weight 8g of wheat germ and transfer into
keep the nuclear membrane intact
beaker, add 30ml detergent to beaker
2. To remove insoluble particles
2. Use funnel and three layers of cheesecloth to
3. To pellet nuclei
50 ml plastic tube
3. Centrifuge the filtrate
4. Pour the supernatant and wash the nuclei
using nuclear isolation buffer
5. Add nuclear isolation buffer to 50ml mark
6. Aliquote 1ml of the wheat nuclei into 1
1.5ml centrifuge tube(lable initials)
7. Place the tube into centrifuge for 1-2 min at
7. remove insoluble contaminnats and
3000rpm
clean nuclei
8. Pour out the supernatent, add 400ul of DNA
incubation buffer to the pellet, pipetting the 8. buffer prepares nuclei to be releases
pellet with P1000 up and down
9. in order to perform 2 protocols
9. Add 600ul DNA incubation buffer to
centrifuge tube, mix and split sample to two 10. SDS breaks the nuclear membrane
tube: 500ul for each and denatures protein, unfold and
10. Add 400ul DNA incubation buffer, 100ul dissociates protein from DNA. NaCl
5M NaCl, 35ul 20% SDS (invert mix) dissociate the histones
11. Water bath the tubes in 60 degree celcius
water for 10 min 11. heat denature proteins
12. Centrifuge both tubes for 3 minutes at
12. pellets the DNA and leave DNA
14000rpm (synchronize wih other students
supernatant
at bench and balance tubes)
13. Pipette 500ul supernatant to 1.5ml 13. supernatant contains proteins
centrufuge tube for both tubes (tube 1: 18
basic protocols, tube 2: 14 refined protocol) 14. remove protein
14. Add 200 ul protein precipitation solution
into supernant tube
15. Vortex the tube for 20s 15. ensure throughout mixing
16. Centrifuge at 14000 rpm for 10 min to
16. protein form a pellet, DNA is in the
solidify the pellet
supernatant
17. Take 300ul from middle part of the tube to
not extract the precipitates and add to a new 17. collects purified DNA
labled tube
18. Add 2 volumes(400ul*2) of ice cold ethanol 18. water forms hydrogen bond with
to precipitate to precipitate the DNA and ethanol instead of DNA, expose the
invert 10 times DNA backbone
19. Centrifuge the tube for 3 min at 14000rpm
(do not lose the pellet). Blot tube dry by
turning it upside down and press
 pellet the precipitated DNA and remove
ethanol to waste the rim of the tube onto 19. compare pellet’sm size: pure DNA is colourless.
paper towel Compare how different protocols affect DNA
20. Redissolve the pelleted DNA in TE buffer, extraction efficieincy
use 100ul of TE to resuspend the pellet
20. prepparation for electrophoresis
produced with basic protocol, use 50ul of
TE for redined protocol, vortex 5-6 seconds,
pipette up and down to r5esuspend the
pellet, put tube on ice while wait.

Agarose Gel Electrophoresis of Wheat Germ DNA

1. Know how to make agarose gel (p10, 19-20)

2. Add 10 ul of pellet just resuspended, 3ul of 2. allow DNA to fluorence undre UV
leading dye, 1ul of 10XSYBR safe DNA
stain to one 1.5ml centrifuge tube
3. Close tube cap and tap tube bottom to mix
4. Each lead 1 lane in the designated gel with 1
wheat germ DNA sample
5. Use micropipettor to lead entire contents of
tube that has wheat germ DNA 14ul into the
separate well in the gel/ per student
Prevent contamination between
a. Use fresh tip
differnet samples
6. Close the top of the elctrophoresis box and
electrical lead (anode-anode, cathode-
cathode)
7. Set to 180 volts
8. Electrophorese in 1.0% DNA agarose gel at
Electrical current allow DNA fragment
180V for 10 minutes
to move and separate by size
9. Good migration of wheat germ DNA:
bromophenol blue band move a short
distance from well to end of gel,m stop
electrophoresis before band run off
10. Turn of power supply and remove top of
electrophoresis box
11. UV transilluminator and observe the
flourescent bands
12. View gel on UV transilluminator and sketch
the gel (does the brightness or intensity of
DNA band gel fit )
 7. stir the MM tube gently with
pipettor
Lab 2: Part 1 protcol: PCR of Orchid 8. aliquote 23ul of MM to clean
DNA 600ul PCR tube

1. set up 1 tube using unkonw

Dna sample, other tube is
negative control: no DNA
2. label the lid of 600ul PCR
tube (UA/UB), label the lid of N is for checking
second PCR tube is negative contamination. If a band
control (N), put initials on appear in this tube in lab 3
each tube then reagent is contaminated
3. making the “master mix”: by stray DNA
Column 5 is the volume of
each reagent that is needed
to pipette to make MM.
a. add 2ul of DNA or
water to PCR tube, 23ul
of master mix so final
PCR volume is 25ul.
Calcul how much water
is needed to make each
tube up to 23ul
(=12.5ul)
4. place all reagents and DNA
on ice Prevent degradation: Taq Dna
a. double check if polymerase and dNTPs are
obtained DNA is correct heat sensitive.
and have a complete
set of PCR reagents (1
MM tube/ student)
5. pipette the required volume
of each reagent into 1.5ml
centrifuge tube (label master
mix);
6. add enzyme: TA pipette the
required amount of Taq DNA
polymerase to MM tube
 do not shake: miminize
number of bubbles as it can
denature proteins and
interfere with the PCR
process

9. add 2ul of unknow DNA to

UA/UB, and 2ul of water to N
tube Added 2ul of DNA at
a. total volume per tube concentration of ~1ng/ul so
should be 25ul there is ~2ng of template
10. keep PCR tube on ice DNA in tube
before use the centrifuge
11. pulse spin: press the
black button for about 1
second. Short spin ensures the
12. thermal cycling: place reactions are thoroughly
tubes on ice until class is mixed and contents are at
ready. TA run thermal cycler the bottom of the PCR tube v
for 36 cycles or specific
temperatures for Reagents must be at the
Paphiopedilum spicerianum bottom: Thermal cycler heats
13. store the completed the bottom of the tube if
reaction at reagents are at the lid or
-20 degree celcius wall, they won’t reach the
required temperature for
denaturation, annealing or
extension.
 Lab 3 Part 2

1. Set up 1 enzyme digest, get PCR tube from a. Add 5ul of undigested PCR to
lab (uknown DNA only) uncut A/B
a. Do not perform on negative control b. Add 5ul of undigested PCR to
b. Each student prepare one enzyme negative control
digest 5. Add 1ul of loading dye to each of these
2. Lable 2*1.5ml centrifuge tubes 1.5ml tubes
a. 1 tube containing UA digested with
Restriction enzye cut DNA at specific sequences
BamHI (UA-bam)
so can analyze fragment size
b. 1 tube containing UA digested with
HindIII (UA-Hind)
c. Label with initials and code
3. Add 5ul of 2X universal restriction buffer to
each tube
a. Pipette tip touch side of tube as
near to bottom as possible Two enzymes: cut at different site: different
4. Add 4ul of PCR prouct from UA tube to fragment patters
each 1.5ml tube
a. Add PCR product directly to
buffer: tip is in the buffer before Provide correct pH, salt, and ions for enzyme
release the UA activity (create pillary action to pull solution out
5. Use P10, add 1ul of HindIII/BamHI to tube of the end of the pipette tip, always add buffer to
a. Do not introduce bubble tube before adding enzyme)
b. Use fresh tip everytime
6. Mix reagents by pulsing in a microfuge/ by Supplies DNA substrate for digestion
sharply tapping the tube bottom on lab
Prevent DNA from sticking to tube walls
bench
7. Place tube in 37 degree celsius water bath
a. incubate for 5-15min, place on ice
Reduce enzyme contact with DNA/ prevent
Part 3 contamination

1. Get 1.0% agarose gel: has at least 20 wells, Evenly mixed

each bench run one gel
2. Add 3ul of loading dye to each digesting
tube Optimal temp for restriction enzyme activity/ ice
a. Place these tubes on ice until step 4 stop enzyme activity
3. Label two new tubes
a. “uncut A/B” & N(negative
control)from PCR
1. Separate DNA fragmens by size
4. Place 5ul of distilled water to each of these 2
2. Add coor so samples are visible
tubes
a. Sucrose so DNA sink into wells
 a. Sketch gel, estimate size of
fragment and label bands
3. Uncut shows origianl fragment size & N
shows no contamination
4. Adjusts volume so samples load evenly

5. Allow proper loading and tracking

6. Add 1ul of loading dye to each of these

1.5ml tubes
7. Mix by pulse spinning
a. Each pair should have 4 * 1.5ml
tubes to lead on agarose gel
i. 2 tubes: restriction enzyme
digest of PCR product
ii. 1: uncutA
iii. 1: negative control
b. Make sure tubes are placed in a
balanced configuration of rotor 8. Prevent DNA contamination
8. Put on gloves
9. Load 11ul total from each tube into separate
wells
a. Skip left most lane: for DNA ladder
10. Load samples in this order
a. Negative control
b. Uncut PCR producte
c. HindIII digest
d. BamHI digest
11. prevent air bubbles
11. Dip pipette tip through surface of burffer
12. Center it over well 12. avoide puncturing gel
13. Depress pipettor to expel sample
14. Load 10ul of loading dy solution containing 13. sucrose in the loading dye weighs down the
DNA ladder to left most lane on both top sample, cause it to sink to the bottom of well
and bottom half of gel 14. ladder provides know fragment sizes: use to
15. Close top of elctrophoresis box estimate unknown fragment lengths
16. Connect electrical leads to a power supply
a. Anode to anode(red), cathode to 15. DNA is negatively charged: move toward anode
cathode (black)
17. Turn power supply on and set to 180V
18. Electrophorese for 15min 18. prevent loss of DNA fragments
a. Stop before bromophenol blue
bands run off end of gel
19. Turn of power supply and give it to TA
20. View gel on ultraviolet transilluminator
20 make DNA visible through fluorescent stain
 roatte the ocular lens to align the stage and
ocualr reticle
3. Calculate lengthof stage micrometer
4. Use age micrometer to measure the total
length of 50 divisions of the ocular reticle
5. Use the recorded value to calculate the um
represent by single division of ocular reticle,
with 10X

Lab 4 : Part 1

1. Use 10X, place specimen slide

a. Turn scope power and adjust light
level
2. Adjust the distance between the two ocular too much light will reduce contr5ast, too littloe will
lenses until obtain bionocular vision not properl illuminate the specimen
a. Never have to close one eye to look
confortably into the microscope
3. Use the focus adjustment knobs to focus the
specimen
a. Start with the stage in high position
and focus away from objectievs
b. Begin with corse adjustment kno
and finish with the fine adjustment ensure that never ruin a slide or damage objective
knob

Adjuste for Koehlar illumination

1. Open the field iris diaphragm and the

condenser iris diaphragm all the way
2. Move the condenser up close to the slide
using the condenser focus knob
3. Close the field iris diaphragm
a. Should see small circle of light
when look at specimen
b. Rotate the condenser focus knobs
until the edges of field are sharply
focused
4. Gradually open filed and stop as soon as the
diaphragm moves out of your field of view
help reduce scattered light and improves
Stage micrometer contrast

1. Calibrate ocular reticle sclae using stage

micrometer of known length
a. 1mm in length, 100 equal units
2. Replace specimen slide with stage
micrometer slide, focus on it using 10X,
 6. Swithc to 40X objective and repeate the
measurement of ocular reticle
a. Calculate number of represented
um

Part 3: paramecium digestion

1. Pipette 20 ul of Paramecium culture into

depressionh slide
2. Mix Congo red yeast cell slury by flicking
the tube
a. Pipette 4ul of yeast on Paramecium
on slide
b. Mix gently with yellow pipette tip Allow Paramecium to feed for 1-2min
3. Pipette 2*20ul of quieting solution and mix Quieting solution is thick and will slow the
with pipettor tip paramecium to allow easier obserbation
a. Avoid transferring bubbles [Link]
your slide by pipetting slowly
4. Place coverslip on slide
a. Slowly lower on e sid of coverslip
over sample
b. Do not trap bubbles
5. Observe paramecium and record what is
observed
6. Draw the single paramesium