Molecular

Biology Laboratory Giulia Ruben

BIOL101L Spring 2017

Lab 1: Basic Lab Skills

HAZARD INFORMATION
Lab coat must be worn whenever you or your lab partner are working in lab; always
wear gloves when you are handling any materials, equipment, or using your pipettors
Amido black stain and de-stain = TOXIC
Use gloves when handling stain and de-stain
Use gloves when handling nitrocellulose & Whatman paper
If you ever suspect your gloves have become contaminated CHANGE THEM

Exercise 1: Assay of Contaminating Growth on Petri Dishes
1. Each group of lab partners will have or obtain a petri dish containing an enriched
bacterial growth medium, Luria broth (LB) and agar.
2. Label this dish on the bottom (why on the bottom?) just near the edge (why?) with your
initials, the date, and your section; label this dish uncovered.
3. There will be another petri dish for each bench; someone at your bench should label this dish
covered.
4. Remove the lid on the dish labeled uncovered and leave it uncovered and open
throughout the lab period; keep the bench dish covered.
5. Before you leave, put the lid back on the uncovered dish and place it, upside down, on the
designated tray on the side bench. (The covered dish for your bench should also be placed
on the tray.) We will incubate them, upside down (why?) for you at 37 deg C. overnight then
store them at 4 deg C.
6. Next week you will examine these plates for growth of microorganisms.


Before you proceed with exercise 2 or exercise 3, your TA must check that
you are using the pipette properly. Once your TA has approved your
technique, you can continue.

Exercise 2. Gravimetric Calibration of Micropipettes and Determination of
Micropipetting Accuracy and Precision

For EACH of the 3 micropipettes (P-20, P-200, P-1000), EACH student should:
1. Set the volume somewhere in the middle of the range of the pipette (e.g. 10 ul for the P-20,
100 ul for the P-200, and 500 ul for the P-1000.)
2. After taring a weigh boat, pipette out that set volume of H20 into the weight boat and
determine the mass of H2O you actually pipetted. Use the density of H20 to determine the
volume you pipetted. Be sure to tare the weight boat on the balance for each weighing!
3. Repeat step #2 a total of 5 times for each pipette.


 4. For each pipette, use the 5 replicate actual volume measurements to calculate:
A. Accuracy = % error
= [(mean of actual volume measurements set volume)/set volume] x 100
B. Precision = S.D.

x = each actual volume measurement
x = arithmetic mean of repeated actual volume measurements
n = number of repetitions
= Sum of

5. In your lab notebook, compare your results to:

A. The performance specifications of the manufacturer (see last page of this handout.) If
your accuracy or precision are well outside the recommended ranges, you probably
need to work on your pipetting technique.

B. Also compare your results to your lab partners results for the same set of
micropipettes; explain and/or speculate about reasons for any discrepancies
between you and your partner's results.

Note: It is a good idea to periodically check the calibration of your micropipettes by
checking their accuracy and precision gravimetrically.

Exercise 3. Bovine Serum Albumin (BSA) Spot Test

1. Prepare BSA Dilutions:
From the stock 5 mg/ml BSA solution you are given, prepare a dilution series of BSA
standards in 5 glass or microcentrifuge tubes (be sure to label the tubes) according to
the scheme outlined in Table 1 below. Be sure to attach a new pipette tip to the
micropipette each time you make a new dilution (why?)

Each lab partner should do his/her own complete set of dilutions.

Table 1. Serial dilution scheme

Tube # Dilution BSA concentration
1 12.5 l of stock (5 mg/ml) + 37.5 l dH20 1.25 mg/ml
2 25 l from tube 1 + 25 l dH20 625 ug /ml
3 25 l from tube 2 + 25 l dH20 312.5 ug /ml
4 25 l from tube 3+ 25 l dH20 156.25 ug /ml
5 25 l from tube 4 + 25 l dH20 78.125 ug /ml



(continued on next page)
2. Spot Test:
We will be using amido black, a stain that quantitatively binds protein (e.g. BSA.) You will
use a micropipette to deliver 2 ul of each of the BSA serial dilutions and a separate spot
of 2 ul of H2O onto the nitrocellulose; you will then stain the nitrocellulose with amido
black. This will enable you to visualize the relative protein amounts in each sample and
provide visual feedback on your pipetting and dilution techniques.

Procedure:
1. Check that you have rectangular piece of nitrocellulose, which should be in a large Petri
dish on your bench, sitting on a round piece of 3MM Whatman filter paper (Whatman,
Clifton, NJ). You and your partner will share the same piece of nitrocellulose.
2. Each student should pipette out a total of 6 spots in a row on the nitrocellose; one spot
should be 2ul of H20, then a separate 2 ul spot of each of the five BSA dilutions. One
partner should spot a top row with his/her samples, and the other partner should spot
a row directly below on the same rectangle of nitrocellulose so you can compare them.
Spotting of the 2 ul is best done by holding the pipette tip just above the nitrocellulose.
Expel liquid such that a drop forms on the end of the tip. Touch the drop to the paper
and the liquid will be drawn into the nitrocellulose by capillary action.
CAUTION: Make certain you leave enough room between each spot such that the spots do
not touch each other, but close enough so that you can fit all 6 spots on the same row.
3. Place the nitrocellulose on a piece of 3MM paper and allow to air dry.

STEPS 4 & 5 SHOULD BE DONE WITH GOGGLES ON
4. After the spots have dried completely, stain the spots by placing your blot in the provided
tray in the fume hood, the tray that has amido black staining solution. Allow to stain for 1-2
min, with gentle shaking in the hood.
5. Remove the blot from the stain and then transfer to another dish with the methanol-
acetic acid destaining solution and shake gently. Transfer again after 5 minutes to
clean destaining solution and shake gently for several more minutes until the
background is white.
6. Remove your blot from the final destain and place it on some 3MM paper to dry.
7. Compare the intensities of each spot. Do the intensities of your spots match your lab
partners? Does each spot appear to be half as intense as the last? Describe what you see
in your lab notebook. If your partner and your spot tests look different or if the spots in
your row dont appear half as intense as the previous, speculate as to the reasons you got
such results. How confident are you in your pipetting technique? As with the calibration
test, the results might indicate that you need to practice your pipetting technique.

BEFORE YOU LEAVE: CLEAN UP
NEVER THROW AWAY STICKER-LABELED TUBES! Sticker-labeled tubes should be put
in rack at TA bench, EXCEPT leave BSA and H20 tubes at your bench for the next section
Do not throw away circular Whatman paper; leave at your bench
Label your coat with lab stations colored tape
Make sure to place your lab coat on your lab days rack
Sign drawer sheet and leave at station
Contamination assay plates: place on cafeteria tray on TA bench to be incubated overnight
Your station must be checked and OK'ed by your TA before you leave
RAININ CLASSIC MICROPIPETTE PERFORMANCE SPECIFICATIONS
(from Rainin micropipette manual)