Expected Results: Zero contamination throughout the whole lab procedures and a successful

gene clone.

Lab 3.A part 4 & 5(Montse)

Materials:
Autoclave(1)
Balance(1)
Weigh boat(2)
100ml graduated cylinder(1)
25ml flask(1)
100ml reagent bottle/flask(1)
60mm petri dishes(4)
100mm petri dish(1)
Heat resistant gloves(1 pair)
Foil and plastic wrap(1 roll each)
Laboratory pen(1)
Distilled water(dH2O)(250ml)
LB agar powder or capsules(6g)
PPE: Safety Goggles. Heat resistant gloves
Safety Concern: Wash hands before and after lab
Making LB Agar
1. Calculate the amount of LB agar required based on the number and size of the petri plates
to be poured. It is not advisable to make less than 50ml of media
2. Label an appropriately sized bottle or flask with the name of the medium, date of
preparation, and initials of the individual preparing by the medium.
3. Measure the required volume of the dH2O and pour it into the labeled container
4. If using LB agar powder, add the required amount of powder. While swirling, slowly add
the LB powder to the dH2O, swirl until no clumps exist. If using LB agar capsules, add
the capsules directly to the water.
5. Place a cap very loosely on the bottle. Steam must be allowed to escape but too much
evaporation will increase the concentration of the agar. Microwave the agar on a medium
setting in 30 sec increments for 1-2 min until it's clear. DO NOT use foil in the
microwave.
6. When the medium is clear, loosely place a cap on the container or foil over the opening.
Put a piece of autoclave tape on the lid of the container, ensuring that the lid is loose.
Place the container into the autoclave
7. Run the autoclave on the liquid cycle once all student groups have placed their media in
the autoclave. Cap the bottles tightly once the media has cooled down.
Pouring Agar Plates
 1. Label the bottom of the petri plates with the name of the medium, date of preparation,
and initials of the individual pouring the plates. Write the label around the outer edge of
the plate
2. Retrieve the autoclaved agar
3. If the agar solidifies before the plates are poured, loosen the cap in the container and
microwave the agar for 1-2 min on medium power, checking every 30 sec until the agar is
completely melted and clear with no lumps. DO NOT use foil in the microwave.
4. Before pouring the plates, cool the molten LB agar to around 55 C so that the container is
comfortable to hold. The molten agar can be left to cool either on the laboratory bench or
in a water bath set to 55 C. Don't let the agar cool so much that it starts to solidify. If this
happens, remelt the agar.
5. Before pouring the plates, gently swirl the agar to mix. Swirl very gently and take care
not to introduce bubbles into the medium.
6. Lift the petri plate and hold it over the plate as a shield while pouring the LB agar into the
plate. Pour enough agar to cover enough(about half of the bottom of the plate) and then
swirl the agar to cover the whole plate. Make sure the plate is about ⅓ full. Replace lid.
7. Pour the remaining plates and allow the plates to solidify undisturbed on the laboratory
bench.
8. Dry the plates on the lab bench for 2 days. Then wrap them in plastic and shore them
upside down at 4 C. The plates should be used within 2 months.

Making LB/Amp/Kan Agar (Val)

- Lab 3A(pt.7 and 5)
PPE: Lab Goggles
Safety Concerns: Make sure to wear protective gloves when handling hot agar
Materials:
➔ 60 mm petri dishes (4)
➔ Heat-resistant gloves (1 pair)
➔ Laboratory tape (1 roll)
➔ Foil (1 roll)
➔ Plastic wrap (1 roll)
➔ Laboratory marking pen (1)
➔ Distilled water (250 ml)
➔ LB agar powder or capsules (6g or 3 capsules)
➔ LB broth powder or capsules (1.25g or 1 capsule)
Techniques: Pouring Agar Plates, Making LB Agar, LB/Amp Agar and LB/Amp/Ara Agar
Plates Concurrently
Procedures: Part 5: Pouring Agar Plates
 1. Label the bottom of petri plates with the name of the medium, date of preparation, and
initials of the individuals pouring the plates. Write the label around the outer edge of the
plate.
2. Retrieve the autoclaves agar
3. If the agar solidifies before the plates are poured, loosen the cap on the container and
microwave the agar for 1-2 min on medium power, checking every 30 sec until the agar is
completely melted and clear with no lumps. Do not use foil in the microwave.
4. Before pouring the plates, cool the molten LB agar to around 55°C so that the container is
comfortable to hold. The molten agar can be left to cool either on the laboratory bench or
in a water bath set at 55°C. Do not let the agar cool so much that it starts to solidify, if
this happens, remelt the agar.
5. Before pouring the plates, gently swirl the agar to mix, swirl very gently and take care not
to introduce bubbles’ into the medium.
6. Lift the lid of a petri plate and hold it over the plate as a shield while pouring the LB agar
into the plate. Pour enough agar to cover about half of the bottom of the plate and then
swirl the agar to cover the whole plate. Make sure the plate is about one-third full.
Replace the lid.
7. Pour the remaining plates and allow the plates to solidify undisturbed on the laboratory
bench.
8. Dry the plates on the laboratory bench for 2 days. Then wrap them in plastic and store
them upside down at 4°C. The plates should be used within 2 months.

Part 7: Making LB Agar, LB/Amp Agar, and LB/Amp/Ara Agar Plate Concurrently
1. Calculate the amount of LB agar required based on the number and size of the petri plate
(and, if making, the number of LB agar deep tubes) to be poured. It is not advisable to
make less than 50 ml of media.
2. Follow the instructions in part 4 to make molten LB agar.
3. Label the outside bottom of 60 mm petri plates with the date, your initials, the name of
the medium, and the supplements added to it (for example, LB/LB amp/ara). Write the
label around the outer edge of the plate.
4. If the agar solidifies before the plates are poured, loosen the cap and microwave the
container for 1-2 min on medium power, checking every 30 sec until the agar is
completely melted and clear with no lumps.
5. Before pouring the plates, cool the molten LB agar to around 55°C so that the container is
comfortable to hold in your hand. Ampicillin is destroyed at temperatures about 60°C.
6. Follow part 5 to pour the LB plates.
7. Estimate the volume of the remaining LB agar using the volume measurements on the
flask or bottle. Using sterile technique, add the appropriate amount of 10 mg/ml (200x)
ampicillin for a final concentration of 50 ug/ml (1x) ampicillin. Swirl gently to mix the
ampicillin with the LB agar and try not to introduce bubbles into the solution.
 8. Pour LB/amp plates
9. Estimate the volume of the remaining LB/amp agar. Using sterile technique, add the
appropriate amount of 200 mg/ml (100x) arabinose for a final concentration of 2 mg/ml
(1x) arabinose. Swirl gently to mix. Pour LB/amp/ara plates
10. Leave the plates undisturbed on the bench to allow the agar to solidify.
11. Dry the plates on the laboratory bench for 2 days. Then wrap them in plastic and store
them upside down at 4°C. LB, LB/amp, and LB/amp/ara agar plates should be used
within 2 months.

Ligation

Safety/Aseptic Techniques:
Disinfect lab bench before and after lab
Keep microtubes closed until ready to use
Change micropipet tips to prevent cross contamination.
Avoid being burned while using the hot water bath by using tongs
Make sure the centrifuge has stopped spinning before opening and removing your tubes.

Materials:
0.5-10 micropipette and tips
Hot water bath
centrifuge

Steps:
1. Heat inactivate restriction enzymes in your pAMp and pKAN tubes by placing them in a
hot water bath @65C for 20 minutes.
2. Label one tube pAMP/pKAN
3. Into this tube transfer the following:
- 4ul of digested pAMP
- 4 ul of digested pKAN
- 1 ul of ligase
- 1 ul of ligase buffer
4. Pulse spin/Centrifuge for 30 seconds to collect all ingredients at the bottom.
5. Leave at room temperature on the lab bench overnight.

Streaking Plates (Amelia)

- Lab 3C(pt. 1b #8)
PPE: goggles and gloves
Safety Concern: Wash Hands before and after lab, being careful near the open flame, and
also be cautious of the bacteria being streaked on plates.
Items-
1. Bunsen burner (1)
2. Inoculation loop (1-3)
3. Sterile toothpick or micropipette tip(1)
4. Microbial Waste container(1)
5. 60mm LBS agar plates(4)
6. Lab marking pen(1)
Protocol-
a. Flame the metal loop by passing it back and forth in the bunsen burner. Let the loop cool
down. Dip the loop into the e.coli HB101 obtain a film of sample across the loop.
b. Gently rub the loop back and forth across the top left corner of the plate about 10 times.
Stay in the top left quadrant of the plate and do not break the surface of the agar. Flame
the loop again.
c. Rotate the plate 45 degrees and draw the loop through one end of the first steak and run
back and forth in the second quadrant about 10 times. Flame the loop again.
d. Rotate the plate 45 degrees and draw the loop through one end of the second streak and
rub the loop back and forth in the third quadrant about 10 times. Flame the loop again.
e. Repeat the process one final time in the fourth quadrant. Flame and cool the loop after
completing streak plating.

Set up the Digestion Reaction (Jacy)

- How to Set Up a Restriction Digest:
- 1. To set up a restriction digest, the components and quantities need to be
determined. Calculate the necessary volume of DNA. This depends on the
concentration of the DNA sample. For example, if 1 ug of DNA is to be digested
in a 20 pl reaction volume and the DNA stock is O.1 ug/ul, then 10 ul of DNA
should be used.
- 2. Calculate the amount of restriction enzyme required. This depends on the
concentration of the enzyme, which typically is printed on the enzyme tube label.
In a digest, 10 U of enzymes are commonly used per ug of DNA. For example, if
the enzyme concentration is 10,000 U/ml, then 1 ul of enzyme contains 10 U, and
1 ul of enzyme should be added to the reaction. Note: When working with DNA
and restriction enzymes, use a fresh, clean pipet tip for each addition to avoid
contaminating the DNA sample with enzymes or the enzymes with DNA.
- 3. Determine the type and volume of restriction digestion buffer. Most restriction
buffers are provided at a 10x concentration and are generally packaged with the
enzyme. If a digestion using two enzymes is to be performed, look up the
manufacturer's recommendation to determine the appropriate buffer for your
application.
 - 4. Calculate the amount of water required to bring the reaction up to the final
volume.
- To set up the reaction, add the calculated volume of the components to a
microcentrifuge tube in the following order: water, buffer, DNA, and, last,
enzyme. When possible, always keep enzymes on ice. Mix the components by
pipetting up and down or gently flicking the tube, and pulse-spin the tube in a
microcentrifuge to collect the contents at the bottom. Incubate the reactions at
37°C in a water bath or dry bath for 30-60 minutes. Place the tube at 4°C until
analysis on an agarose gel.
- Materials:
- (1) Water bath, dry bath, or incubator at 37°C (shared)
- (1) Microcentrifuge or mini centrifuge (optional) (shared)
- (1) 2-20 ul adjustable-volume micropipette and tips
- (4) Colored microcentrifuge tubes (yellow, violet, green, and orange)
- (1) Microcentrifuge tube rack
- (1) Floating tube rack (optional)
- (1) Container of ice
- (1) Laboratory marking pen
- (1) Waste container
- (25 ul) Lambda DNA on ice
- (60 ul) 2x restriction buffer on ice
- (5 ul) EcoRI enzyme on ice
- (5 ul) Hindill enzyme on ice
- (5 ul) Pstl enzyme on ice
- Procedures
1. Label colored microcentrifuge tubes as below:
- yellow tube - L (lambda DNA)
- violet tube - P (Pstl lambda digest)
- green tube - E (EcoRI lambda digest)
- orange tube - H (Hindill lambda digest)
Label all the tubes with your initials and date, and place them in the
microcentrifuge tube rack
2. Using a fresh tip for each reagent and sample, pipet the lambda DNA, 2x
restriction buffer and enzymes into each tube according to the table below. Make
sure the reagents are added in the order indicated: DNA, followed by buffer, and
last, enzyme.
Tube DNA Buffer Pstl EcoRl Hindlll

L 4 ul 6 ul - - -

P 4 ul 5 ul 1 ul - -
 E 4 ul 5 ul - 1 ul -

H 4 ul 5 ul - - 1 ul

3. Tightly cap the tubes and mix the components by gently flicking the tubes with
your finger. Collect the sample at the bottom of each tube by tapping the tube
gently on the bench or by pulse-spinning it in a microcentrifuge.
4. Incubate digestion reactions for 30 min at 37°C or overnight at room temperature.
5. After the incubation, store the samples at 4°C until the next laboratory period.
Samples can be stored for 1 month at 4°C. If there is sufficient time, proceed to
running the gel.
6. 1x TAE electrophoresis buffer and a 1% agarose TAE gel are required for the next
part of the activity. If necessary, prepare 1x TAE (refer to part 1 of Activity 2.D)
and a 1% agarose TAE gel (refer to Activity 4.B).

Bacterial Transformation with pKAN/pAMP Plasmid (Mayra)

- Lab 5B (starts page 167)
1. Items
a. 1 Incubator at 37 degree celsius (shared)
b. 1 Water bath at 42 degree celsius (shared)
c. 1 2-20 ul adjustable-volume micropipette and sterile tips
d. 1 20-200 ul adjustable-volume micropipette and sterile tips
e. 1 100-1000 ul adjustable-volume micropipette and sterile tips
f. 2 Sterile microcentrifuge tube rack
g. 1 Microcentrifuge tube rack
h. 1 Floating microcentrifuge tube rack
i. 1 Bunseen burner
j. 1 Inoculation loop
k. 1 UV light
l. 1 Parafilm (shared)
m. 1 roll laboratory (optional) (shared)
n. 1 Container of tube
o. 1 Laboratory marking pen
p. 1 Microbial waste container
q. 600 ul Transformation solution (0.05 M CaCl2)
r. 10 ul pKan plasmid (0.08 ug/ul)
s. 1 ml LB broth
t. 2 LB agar plates
u. 2 LB/amp/kan agar plates
v. 1 LB agar starter plate with E. coli (HB101) colonies from part 1
2. Procedures
a. If necessary, 3 days prior to the activity make 2 LB plates (label one positive and
the other negative), 2 LB/amp/kan (one positive and the other negative)
b. One day prior to the activity, streak one LB agar plate for single colonies with E.
coli HB101 bacteria (see step 8 of Activity 3. C). Incubate the plate overnight at
37 degree celsius. This is the starter plate for step 5.
c. On the day of the activity, label one microcentrifuge tube +pKan and another
-pKan. Label both tubes with your initials
d. Using aseptic technique, pipet 2 ul of transmission solution (0.05 M CaCl2) into
each tube. Place both tubes with your initials.
e. Flame the inoculation loop and let it cool down. Scrape 2-4 single E. coli colonies
from the surface of the starter plate so they collect on the loop. Transfer the loop
into the +pKan tube and swirl it in the transformation solution to disperse the
bacteria. Close the tube and place it back on ice.
i. Note: A sterile plastic inoculation loop can also be used to pick the
colonies but should not be flamed
f. Flame the loop and repeat step 5 to transfer bacteria to the -pKan tube.
g. Pipet 10 ul of pKan plasmid into the +pKan tube. DO not add any plasmid into
the -pKan tube. Mic by pipetting gently up and down
h. Incubate the tubes on ice for 10 min, making sure there tubes are in full contact
with the ice.
i. Label two LB agar plates, two LB/amp/kan with your initials and the date. Label
the plates as follows:
i. LB +
ii. LB -
iii. LB/amp/kan +
iv. LB/amp/kan -
j. After 10 min, transfer the +pKan and -pKan tubes directly from the ice into the 42
degree celsius water bath for exactly 50 sec, then immediately place them back on
ice. Incubation at 42 degree celsius is used to heat shock the bacteria.
i. Note: Make sure the tubes are in full contact with the 42 degree celsius
water. For the best results, do not exceed the incubation time on the ice
and at 42 degree celsius water.
k. Incubate the tubes on ice for 2 min
l. Remove the tubes from the ice and pipet 250 ul of LB broth into each tube using
sterile technique. Incubate the tubes at room temperature for 10 min.
m. Mix the tubes by inverting. Pipet 100 ul of each transformation mixture into the
appropriately labeled LB agar plate, or LB/amp/kan agar plate using a sterile pipet
tip each time.
 n. Flame the inoculating loop and let it cool down. Spread the bacteria over the
entire surface of the plate in all directions. Flame the loop.
i. Note: A sterile plastic inoculation loop can be used to spread the bacteria
but should not be flamed.
o. Repeat for each plate.
p. Stack the plates together with the agar side up. Incubate the plates in a 37 degree
celsius incubator for 16-24 hr.
q. Once the cultures have grown for 16-24 hr, remove them from the incubator.
Wrap the plates in Parafilm, and store them at 4 degree celsius for up to 2 weeks.
3) Safety Concerns: Wear lab goggles for small risk of splash and be careful with the fire from
the bunsen burner
4) Expectations: Bacteria is transformed and with pKan gene