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BIOL2103
Biological Sciences Laboratory Course
Practical 1

Part A: Laboratory manual

DNA sequencing and analysis
Introduction:
DNA sequencing is a process that allows us to determine the order of nucleotides
that makes up a DNA molecule. You are going to set up a DNA sequencing reaction
using the dye-terminator sequencing approach. A DNA plasmid and a sequence primer
will be provided, but not any clue to what DNA was cloned into the vector. In this
practical, you will set up the cycle sequencing reaction, purify the extension product
using ethanol/EDTA precipitation method and perform sample electrophoresis on the ABI
Prism 3700 DNA analyzer. Thereafter, you can collect your own sequencing result and
analyze the sequence of your unknown plasmid using a program called BLAST (Basic
Local Sequence Alignment Search Tool) to identify what gene or piece of a gene you
have sequenced and then you have to translate your sequenced DNA piece into amino
acid sequence.

Workflow

Time Task Work done by

1:30 - 2:00 pm (i) Reminder on use of pipettmen
(ii) Briefing (Part A of the lab manual)
2:00 - 2:30 pm (i) Prepare sequencing reaction

2:30 - 4:00 pm (i) Briefing (Part B of the lab manual) by demonstrators Student
(ii) Download Chromas 2 / 4Peaks
(iii) Learn to use Chromas 2 / 4Peaks and BLAST
4:00 - 5:00 pm (i) Ethanol/EDTA precipitation
(ii) Load sample into a 96-well-plate

Remember: Bring your laptop computer when you attend the

practical session

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Set up cycle sequencing reaction

Each of you will be provided with a plasmid DNA with a specified number and
remember write down that number in your report. Set up your own cycle sequencing
reaction as shown below.

Add the following into a 0.2ml PCR tube:

BigDye ver 3.1 sequencing mix 4µl
Plasmid DNA (No.: ) 1µl (~200ng)
Sequencing primer (10µM) 2µl (3.2pmol)
PCR H2O 3µl
Total 10µl

Cycle sequencing:
1. 95oC for 1min
2. Repeat the following for 25 cycles:
• 95oC for 10sec
• 50oC for 5sec
• 60oC for 2min 30sec
3. Hold at 4oC

This cycle sequencing reaction can sequence either single- or double-stranded DNA by
adjusting the quantity of template DNA in the reaction.
The template quantity is listed below for your reference.
PCR product *
100-200 bp 1-3ng
200-500 bp 3-10ng
500-1000 bp 5-20ng
1-2 kbp 10-50ng
>2 kbp 50-100ng
Single-stranded 25-50ng
Double-stranded 100-200ng
Bacterial genomic DNA 2-3µg
* PCR product should be purified to remove PCR primer

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pcDNA3.1 vector
MAP

Multiple cloning sites of pcDNA3.1(+) vector

Sequencing primer

Sequencing primer for pcDNA3.1(+) vector (forward primer):

5’-CGG TAG GCG TGT ACG GTG GG-3’

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Ethanol/EDTA precipitation
• To precipitate the extension product

1. Add the following into the PCR tube containing cycle sequencing reaction product
125mM EDTA 2.5µl
95% ethanol 30µl
2. Mix well and incubate at room temperature for 15min
3. Spin at 13000g for 20min
4. Discard the supernatant without disturbing the pellet
(Remark: you may not see, but it should be at the bottom)
5. Wash the pellet with 30µl of 70% ethanol
6. Spin at 13000g for 5min
7. Discard the supernatant without disturbing the pellet
8. Dry the pellet at 95oC for 2min (with the lid opened)
9. Resuspend the pellet in 10µl of HiDi formamide
10. Vortex to resuspend the pellet
11. Load your sample into a 96-well plate

12. Heat denature at 95oC for 2min (done by technician)

13. Stand on ice for 2min
14. Sample electrophoresis

Analyzing your DNA sequence data

In order to learn how to use the software, please bring your own notebook computer in
the practical session. You also need to download the software called Chromas 2
(Windows) or 4Peaks (Mac). Your demonstrator will instruct you how to do so during the
practical session.

There are several DNA databases that maintain extensive networks of information on
cloned genes worldwide. Many of these databases are maintained as free-access sites
on the Internet. The most complete database, called GenBank, is maintained by the
National Institutes of Health. GenBank contains all publicly accessible DNA sequences
(over 9 billion bases to date with thousands of sequences added each week).
When a gene, or a piece of a gene, is cloned for the first time, the gene is assigned a
GenBank accession number which is included when the gene sequence is reported in
the literature. Using this number to search GenBank, it is then possible to obtain detailed
information about the nucleotide sequence of a gene, the protein encoded by the gene,
intron-exon boundaries, information about the investigators who cloned the gene, and
pertinent literature references among many other facts.

You read your sequencing result using Chromas and delete the nucleotide sequence of
the vector backbone and input the remaining sequence into a search tool called BLAST
(Basic Local Alignment Search Tool) to access GenBank via the NCBI site. BLAST
site enables you to search GenBank by entering a sequence of DNA nucleotides. If
there is a sequence in GenBank that is similar or identical to the nucleotide sequence
that you entered, BLAST will give you possible gene matches with percent similarities
between the sequence you entered and possible matches (rank ordered according to
sequences with the greatest similarity).

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Data analysis
Use BLAST as follows:
1. Use your favorite browser software to access the BLAST site at the National Center
for Biotechnology Information:
http://www.ncbi.nlm.nih.gov/BLAST
2. Click the link to “Nucleotide-nucleotide BLAST [blastn].” A page with a search box
will appear. Cut and paste the sequence (remember delete the sequence of the
vector) and enter it into this text box. Click the “Blast!” button. Your results will be
available in a minute or two. Click the “Format!” button to see the results of your
search. A page will appear with the results of your search).
3. The top sequence shown will be the most likely match.

Report
Write a concise report (not more than 5 pages; not including the first page of your
sequencing result and the first page of the blast result) describing your analysis.
In the report, you should include:
• Your plasmid DNA number
• Number of bases you get from your sequencing experiment (exclude the
nucleotide of the vector backbone)
• The gene which your result sequence most likely matched
• Amino acid sequence translated from your result sequence
• Some background information of the gene you identified

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Part B: Basic techniques

(1) Working with liquid

Measuring and dispensing liquids

The equipment you should choose to measure liquids depends on the volume being
dispensed, the accuracy required and the number of the times the job must be
performed (Table 1).

Table 1. Criteria for choosing a method for measuring a liquid

Special notes for handling and measuring certain liquids:

1. High viscosity liquids are difficult to dispense: Allow time for all the liquid to transfer
2. Organic solvent evaporate rapidly and make measurements inaccurate: Work quickly
and seal the containers without delay
3. Solution prone to frothing (e.g. detergent and protein sample):Avoid forming bubbles
due to overagitation and do not transfer quickly
4. Suspension (cell culture) may sediment: Mix thoroughly before dispensing

(A) Pasteur pipettes

Keep the pipette vertical with the middle fingers
gripping the barrel while the thumb and index
fingers apply pressure on the bulb.
Squeeze gently to dispense individual drops

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(B) Measuring cylinders and volumetric flasks

Must be used on a level surface and your eyes are level with the bottom of meniscus
Fill with solution near but below the desired mark. Fill slowly (e.g. using a Pasteur
pipette) until the bottom of meniscus leveled with the mark

Meniscus

It should be read as 6.6 ml,

but not 7.2 ml

(C) Plastic pipettes

Graduated and bulb (volumetic) pipettes
Never mouth-pipette
Using bungs or Pi-pump
Hold the pipette and bung close together when
connecting the pipette into bungs (see below) or
pipette aids

(a, b) Graduated pipettes

(c) Bulb pipette

(D) Weighting
Know the density of the liquid and apply the following equation
Mass/density = volume
Convert the volume to mass and weight the liquid
e.g. Measure 7.5ml of the liquid A (density of liquid A:1.2 g/ml) using a weight balance
Mass/1.2 = 7.5
Mass = 9g

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(E) Pipettors
Standard Operating Procedure for pipettors (Adopted from the user manuals of two
major brands: Eppendorf and Gilson)

Eppendorf pipettors

Eppendorf Research Series, adjustable-volume

To get a brief idea how to use a common type of pipettor (brand: Gilson), please
visit the following website before attending the practical session.
http://www.bio.upenn.edu/computing/media/Instructional.Pipetman.php

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Steps of Pipetting
(1) Set the volume by selecting a pipettor (cover the range) and turning the setting
ring. See the examples below:

P20 P200 P1000

17.5 ul 125.2 ul 950 ul
(2) Fit a new tip onto the pipette
• Attach the selected tip onto the nose cone of the pipette to ensure a firm and
airtight seal
(3) Pre-rinse the tip (Not a must, see special notes)
(4) Aspirate liquid
• Press the control button to the first stop
• Immerse the pipette tips approximately 3mm into the liquid (5mm for 500-5000µl)
and make sure that the pipette remains in fully vertical position
• Aspirate the test volume slowly and wait 3sec before removing the pipette from
the liquid
• Pull the pipette tip from the test liquid slowly
(5) Dispense liquid
• Touch the filled tip against the inner wall of the tube or vessel at an angle of
approx. 30° to 45°
• Press down the control button slowly to the first stop and wait until the liquid
stops flowing
• Press the control button to the second stop (blow-out) until no liquid remaining in
the tip
• Hold down the control button and pull the tip up along the inner wall of the tube or
vessel
• Allow the control button slide back to the original position

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Special notes
• It is important to pre-rinse or pre-wet the tips when pipetting some liquid. Liquids
(e.g. protein-containing solutions, detergent and organic solvents) form a thin film the
inner wall of the pipette tip; pre-wet the tip to minimize any errors that is related to
this phenomenon.
Pre-wetting consists of aspirating the first volume of liquid and then dispensing it
back into the same vessel. Subsequent volumes that you pipette will have levels of
accuracy and precision.
• Pipette liquid volume of <10µl
The specified values for accuracy and precision can only be attained if the volume
from a tip that has NOT been pre-wetted is used.

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