BE/CS 196a Lab 3: Basic lab technique and
measuring the rate of a DNA strand displacement reaction
Note: Download and open in Adobe Acrobat to see the links in this file.
1. Safety training (20 min)
2. Pipetting practice (60 min)
If you have not pipetted before, this is a chance to learn and practice pipetting.
If you have pipetted before, this is a chance to find out how good you are at pipetting.
• Step 1: read the pipetting instructions carefully.
• Step 2: pipet the volumes of water listed in the Excel file into a weighing boat on a
balance, and record the weight of water. Two persons per team, one will pipet while
another records the measurements, and then you will swap.
a) Use small sized weighing boat for 2-200 µL volume, and medium sized weighing
boat for 300-1000 µL volume.
b) Change a pipette tip for each measurement.
c) Repeat each volume until the most recent three measurements are all within the
target weight.
• Step 3: data analysis. Calculate the mean and standard deviation of your last three
measurements for each volume, and then calculate and plot your pipetting accuracy
and precision (in %). Accuracy = (Expected Weight - Mean) / Expected Weight;
Precision = Standard Deviation / Mean. (1 µL H2 O is 1 mg)
Figure 1: Example plot of pipetting accuracy and precision.
• Include the data analysis in your experimental report for Lab 3 and 4.
3. Buffer preparation (20 min)
1
• Step 1: label all vessels that you ever use, including test tubes of 0.5 mL, 1.5 mL and
50 mL volume and glass vials, even if it contains just water. Your label should include
name and concentration (if applicable) of the buffer or sample inside the vessel, your
initial, and the date.
• Step 2: Mix the following ingredients in a glass vial. Note that H2 O always refers to
purified Milli-Q water.
1× TE/10× Mg2+
MgCl2 (1 M)
625 µL
100× TE (pH 8.0)
50 µL
H2 O
4.325 mL
Total
5 mL
4. Measuring the rate of a DNA strand displacement reaction (60 min)
w5,6
S6
S5
Rep6
+
S6
T
T*
S6*
krep
Q (quencher)
F (fluorophore)
S5
T
T*
Fluor6
+
S6
S6*
F
waste
S6
Q
Figure 2: Domain-level diagram of a DNA strand displacement reaction.
• Step 1: Set up a fluorescence experiment on the plate reader (BioTek Synergy H1). Try
synchronizing steps 3 and 4 with your team members who will perform the experiments
using the same 96-well plate.
• Step 2: vortex and spin down each test tube of sample before use.
Instructions for using a mini centrifuge and vortexer.
• Step 3: Mix the following ingredients in four test tubes, then vortex thoroughly and spin
down briefly. The target concentrations are Rep6 = 50 nM and w5,6 = 0/10/20/30 nM.
Check out the protocol for calculating the pipetting volume of each ingredient.
Record the time that you add Rep6, which is when the reactions are started.
1× TE
1× TE/10× Mg2+
20T (500 µM)
w5,6 (5 µM)
Rep6 (20 µM)
Sample 1 Sample 2 Sample 3
447 µL
446 µL
445 µL
49.8 µL
49.8 µL
49.8 µL
2 µL
2 µL
2 µL
0 µL
1 µL
2 µL
1.25 µL
1.25 µL
1.25 µL
2
Sample 4
444 µL
49.8 µL
2 µL
3 µL
1.25 µL
Note: The accuracy with which we can prepare sample concentrations is limited because DNA molecules stick to the walls of pipette tips and test tubes. To minimize
this issue, we use Lo-Retention tips and Lo-Bind tubes. On top of that we use 20T,
a single strand with 20 nucleotides of T’s, at a much higher concentration compared
to other DNA molecules such that it sticks to the tips and tubes more preferably and
thus reduces the loss of other DNA molecules.
• Step 4: Transfer the four samples from test tubes to a 96-well plate (pipet each sample
into 4 wells, with 100 µL per well), and measure fluorescence kinetics using a plate
reader. Record the time when the measurements are started. The delay time between
when the reactions were started and when the measurements were started will be used
in your data analysis.
• Step 5: Data analysis.
a) Plot your raw data as (fluorescence, time).
b) Normalize your data using the fluorescence of Sample 1 at t = 0 as 0 nM and the
average of the last 5 data points of Sample 4 as 30 nM, and plot your normalized
data as (concentration, time).
c) Simulate the strand displacement reaction in Figure 2, with an initial rate constant
of krep = 5 × 104 /M/s.
d) Compare your simulation with data, and find a rate constant krep that fits your
data reasonably well.
Rep6 = 50 nM, krep = 5×104 /M/s
30
Conc . (nM)
25
20
w5,6
0 nM
15
10 nM
10
20 nM
30 nM
5
0
10
20
Time (min)
30
40
Figure 3: Example plot of finding the rate constant of w5,6 + Rep6 → Fluor6 + waste. Lines indicating
simulations and dots indicating experimental data (dots are actually simulations in this plot, your experimental data will be much noisier, and depending on how fast you are, your data will not include the first 5
to 10 min of the reactions).
• Include the data analysis in your experimental report for Lab 3 and 4.
3
5. Cleaning (10 min)
Clean up your bench after each wet lab.
• Store all samples in boxes in the fridge.
• Place all pipettes back in their hangers.
• Cover all pipet tip boxes with their covers.
• Empty your trash bin (there are three large trash cans in the lab, one under bench 1,
one next to bench 3 and 4, one near the entrance).
• Collect your notes.
4