Flowchart 7 “16” or “rpo”.
Place appropriate spin
Name: Yinjia “Ivan” Wang
Lab Station: 9
Reading orientation: Top → Down in each
column. Left column, then right column.
Safety:
- Safety goggles
- Lab coat
- Gloves
Prepare a flowchart detailing the necessary
steps to analyze your PCR Product.
Cleanup
- Label the side of the capless collection
tubes, and lid of the spin columns and
orange-colored BIND tubes with group
number and sample name.
- Use P200 to transfer 50 uL of each of
PCR samples into the corresponding
orange-colored tubes (containing 250 uL
of BIND buffer.
- Use P1000 to everything (300 uL total)
to the center of the appropriately labeled
spin column.
- Balance two samples on the centrifuge.
Centrifuge for 60 seconds at 10k X g.
(DNA will bind to the silica matrix in
the spin column
- Discard flow through into the waste
beaker. Replace columns back into the
empty collection tube.
- Wash spin columns by adding 700 uL of
WASH Buffer (Green colored tube) in
each columns and centrifuge 60s.
- Discard flow-through into liquid waste.
Replace the columns in to the collection
tubes and centrifuge for an additional
minute(remove residual wash buffer).
- Label 2 empty 1.5 mL microcentrifuge
tubes with section number, group
number, and
columns into these tubes. This tube will
be used to collect DNA.
- Dispense 30 uL of ELUTE buffer
(Purple tube) directly to the white silica
matrix in the center of spin columns.
Close the cap of spin column. The
collection tube remains open. Let
columns stand for 1 min and then
centrifuge for 1 min.
- Remove spin columns and sicard them
into the solid waste beaker. Do not
discard the 1.5 mL microcentrifuge
tubes - these contain purified PCR-
amplified DNA frags. Cap and place in
rack at workstation.
Gel Electrophoresis
- Take two clear 0.5 mL tubes and use a
Sharpie to label one “16” and the other
“rpo.”
- Use a P20 micropipette to remove 5 µL
from each of your purified PCR samples
and place it in the appropriately labeled
0.5 mL tube. Do not throw away the rest
of the purified PCR sample, as you will
use this to set up your sequencing
reaction.
- Use a P20 to add 15 µL of the 1.33X
loading dye to the 5 µL of sample you
just pipetted into the 0.5 mL tube.
Gently pipette up and down to mix the
solutions.
- The instructor will load lanes 1 and 12
with 20 µL each of negative control
reactions for 16S and rpoB. The
instructor will also load lanes 3 and 4 of
the Bench 1 gel with positive control
reactions for 16S and rpoB.
- Lanes 2 and 11 will be loaded with 20
µL of a 1Kb ladder DNA solution
(marker DNA of known size and
concentration). The gels should be
loaded according to Figure 5. Maps for
loading the lanes are also next to each
gel.
 - Use a P20 to load 20 µL from each of
your reactions into the appropriate well.
Use a new tip for each reaction. Use two
hands to steady the tip over the well. Be
careful not to punch the pipette tip
through the gel.
- Once the entire gel is loaded, press and
release the 30 minute button to start a 30
minute run. The light will change to a
steady green. After 30 minutes, the gel
automatically shuts off. You will hear a
rapid beeping and the light will flash
red. Press and release the 30 minute
button to stop the beeping.
- Your instructor will collect your gel,
place it in the LED illuminator, and
photograph it. You will use this
photograph to analyze your results. The
SYBR Safe binds to the DNA and
fluoresces when exposed to the blue
light. You will compare the position and
fluorescence intensity of your bands to
the position and fluorescence of various
DNA bands in the ladder DNA. The
expected sizes of your DNA products
are 1,484 bp for 16S rRNA and 1,112 bp
for rpoB.
- Make sure that you get a picture or print
out of your gel image, as you will need
it for your post-lab.
- Set up the two sequencing reaction for
rpoB by solving for the
- ng Estimated DNA Amount
- ng/uL Estimated DNA
Concentration (= i / 5 uL)
- uL needed for 30 ng of DNA
(30 ng / ii ng /uL)
- Label two sterile 0.5 mL centrifuge
tubes with your group number and
“rpoF” (for forward) or “rpoR” (for
reverse). Set up the reactions using the
table below (Table 2).
- Place your sequencing reactions in the
GREEN rack on instructor bench into
appropriate labeled wells. Your
instructor will add 1 uL of appropriate
primer to each tube and transfer the
sample to the BLUE rack. Results come
next week.

Prepare a Flowchart on how to set up your

sequencing reactions once you have the data
from the gel.

16S

- Set up the two sequencing reactions for

16s rRNA sample by solving the
- ng Estimated DNA Amount
- ng/uL Estimated DNA
Concentration (= i / 5 uL)
- uL needed for 30 ng of DNA
(30 ng / ii ng /uL)
- Label two sterile 0.5 ml centrifuge tubes
with your group number and “16F”
forward and “16R” reverse.
- Determine the amount of water required
to reach a final volume of 12 uL for
each reaction.

rpoB