Name_________________________________________
Bio250: Biotechnology in Environmental Systems
The Cooper Union
Laboratory Module 2 of 3:
Design and function testing of a plasmid based biosensor system in the gramnegative bacterium Escherichia Coli (E. coli).
Lab Session 2 / Agarose Gel Electrophoresis and ligation of digested pCDF-Duet-1
Plasmid DNA and amplified GFP DNA.
Objectives
1. Learn how to set up agarose gel electrophoresis for your digested plasmid
DNA
2. Learn how to set up a ligation reaction
Gel electrophoresis background:
The fastest method to quickly assess whether the restriction endonuclease
reaction worked is through gel electrophoresis. Gel electrophoresis uses an electric
current to separate molecules by size and charge through a conductive hydrogel. The
DNA samples are loaded into wells of an agarose gel and electrophoresed.
When the current is applied, smaller molecules will migrate faster than larger
molecules; this is due the pores formed within the gel and the resulting friction of
molecules moving through the gel. The researcher can reduce the pore size by
increasing the concentration of the polymer (for this lab, agarose). In order to view the
products embedded within the gel, a fluorescent stain is added while the gel is molten. In
this lab we will use a dye, Ethidium bromide, that intercalates within the base pairs of
DNA.
To determine which band correlates to the target molecule, a marker is added to
the gel simultaneously. The marker (also known as a molecular weight standard or
ladder), is composed of molecules of known and varying size. By measuring the
distance the known standard traveled through the gel and plotting that data against the
known marker sizes, we can derive the estimated sizes of the sample molecules, in this
case, the digested plasmid DNA or PCR amplified DNA.
You will be using the Kb plus DNA ladder as a molecular ruler, from Goldbio.com (1 kb
PLUS™ DNA Ladder | GoldBio ). See below.
Please watch the two gel electrophoresis videos that are linked on the Bio 250
course Moodle page.
You and your group partners will be loading your samples in the order given below:
1. Gel Electrophoresis Pre-Lab Questions:
Question 1: After you have digested your pCDFDuet-1 plasmid using the restriction
enzymes Sal I and Not I, what is the projected size of the remaining plasmid, minus the
insert that was removed? (FYI, the larger piece containing the origins of replication,
selectable markers and cloning site is referred to as the plasmid “backbone”.)
Question 2. What is the size of the insert that was removed from the plasmid?
2. LIgation Pre-Lab Questions:
Your will be setting up a ligation reaction of the plasmid, pCDFDuet-1, along with the
amplified GFP insert to generate the IPTG biosensor. You will be using the T4 DNA
Ligase enzyme to covalently bond both pieces of DNA together. You will perform the
calculations below in setting up your ligation protocol.
Ligating your amplified GFP insert into your digested pCDFDUET-1 plasmid.
Go to the New England Biolabs website and search for the enzyme T4 Ligase. On the
same website find the “Ligation Protocol for T4 DNA Ligase (M0202)”. Using the
framework of this protocol, write down the exact protocol below for a ligation reaction
below.
Use these values: Total Reaction Volume, 21 uL (including 1 uL Ligase enzyme), 2 uL
10X T4 DNA Ligase Buffer, 50 ng digested plasmid DNA.
Assume that your Sal I/Not I double digested pCDFDuet-1 plasmid (size: 4330 bp or
4.33 Kb) is at a concentration of 100 ng (nanograms) per uL (micro liter) and that your
digested insert DNAs (insert 1, size: 1000 bp or 1 Kb) is at a concentration of 100 ng per
uL.
Assume that your insert:plasmid molar ratio will be 3:1 for each insert:plasmid
combination. Use the handy online Ligation Calculator (http://www.insilico.uniduesseldorf.de/Lig_Input.html) to calculate the exact amount of insert that you will need.
You will need to then convert the oligo insert and plasmid amounts in uL volumes below.
Note that when you set out to do the calculations, the software may indicate
that the amounts you may need to pipette are far less than 0.5ul. Considering that
the accuracy of your pipettors is not adequate below this range, what can you do?
Considering that the concentration of your DNA is sufficiently high, one approach
would be to dilute a sample of your DNA solution to a range that you can accurately
pipette. For example, assume that you have a solution of DNA that is at a
concentration of 200 ng/uL and you are tasked with pipetting 10 ng of DNA in a
total reaction of 20 uL. You wouldn’t be able to practically pipette 0.05 uL (10ng) of
solution. What you could do is instead prepare a dilute “working sample” of your
more concentrated “stock solution” that is at a lower dilution. In this case you could
pipette 1uL of your 200 ng/uL stock solution into 19 uL of water or buffer, giving
you a total volume of 20 uL at a final concentration of 10ng/uL. From this “working
sample” you can now pipette 1uL volume of solution containing 10ng of DNA. For
your protocol below, use this same technique. Assume that you have ample volume
of DNA “stock solutions” (ie. more than 100 uL) to work with.
Question 3. What are the volumes of insert and vector DNA required to start the ligation
reaction? Fill in your answers below:
Reaction Total Volume
21 μl
10X T4 DNA Ligase Buffer*
2 μl
Vector DNA (_____kb)
50 ng (____uL)
Insert 1 DNA (______kb)
_____ng (_____uL)
Nuclease-free water
to 20 μl
T4 DNA Ligase
1 μl
Question 4: Write down the detailed ligation protocol, including exact incubation
temperatures and reaction times, along with your calculations for DNA “working
solution” concentrations: