Southwestern College
Biotechnology Workshop
Instructor: Elmar Schmid, Ph.D.
Ligation Reaction
Necessary Equipment:
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Water purifying system (e.g. Millipore system) to retrieve pure water with doubledistilled quality
Microcentrifuge
Water bath (adjusted to 15oC)
Ice bucket (filled with ice)
Plastic racks for 0.5 ml reaction tubes
Styrofoam float (for 0.5 ml reaction tubes)
Adjustable-volume digital pipettes (e.g. Eppendorf, Finnipette)
Required Materials & Reagents:
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DNAse-free, double-distilled water (= ddH2O)
Your PCR-amplified and gel purified bacterial gene fragment
(your instructor will tell you which PCR amplicon you will be ligating into
which bacterial expression vector)
T4 DNA Ligase (= is part of the PinPoint Xa-1 T vector kit: Promega)
 1 -3 Weiss units/μl
PinPoint Xa-1 T or pIVEX vector
 part of the PinPoint Xa-1 T (Promega) or the
RTS AviTag Biotinylation vector kit (Roche)
 the vectors are usually already linearized; therefore no restriction digest is
necessary as preparation for this lab
PinPoint T-vector Control DNA
 part of the PinPoint Xa-1 T vector kit: Promega)
T4 DNA Ligase 10x Ligation Buffer
 part of the PinPoint Xa-1 T vector kit: (Promega)
 10-times concentrated buffer enabling ideal ligation conditions
Sterile/autoclaved PP reaction tubes (0.5 ml)
Pipette tips (crystal, yellow, blue)
1
Southwestern College
Biotechnology Workshop
Instructor: Elmar Schmid, Ph.D.
Procedure:
a. Make sure that all solutions are thawed and place them in an ice-filled bucket
b. Centrifuge the (closed) vials containing the LINEARIZED bacterial expression
vector, e.g. PinPoint Xa-1 T or pIVEX vector and the control vector (if
applicable, e.g. the PinPoint T-vector Control DNA, briefly (5 – 10 sec) in the
microcentrifuge at max. speed to collect the vial contents at the bottom of the
vial; place them back on ice
c. Set up the ligation reactions by pipetting following solutions and components
into sterile 0.5 ml PP reaction tubes following the Pipette Scheme below
 pipette the components into the tubes starting with water, then
the other ones from top to bottom
 make sure you change the tips in between each pipetting step
 put the DNA and ligase back on ice after you used it
Pipetting Scheme: Ligation Reactions
Tube No.
1
No DNA
insert
(= blank)
[ μl ]
7
2
3
4
5
Control
DNA
[ μl ]
4
PCR
amplicon 1
[ μl ]
10 - x
PCR
amplicon 2
[ μl ]
10 - y
PCR
amplicon 3
[μl]
10-z
10x Ligase
Buffer
Vector DNA
(50ng)
PCR product
1
1
1
1
1
1
1
1
1
1
-
-
x
y
z
Vector
Control DNA
T4 DNA
ligase
(1 -3 units)
Total
Volume
[ μl ]
-
3
-
-
-
1
1
1
1
1
10
10
10
10
10
ddH2O
d. Close-cap the tubes and briefly mix the contents by flicking the tubes with
your fingers
e. Place the tubes into the size-matched holes of a Stryro-foam float and
incubate them in a water bath for at least 3 hours at 15oC
f. Take the tubes out of the water bath and place them in a plastic rack suitable
for 0.5 ml reaction tubes
2
Southwestern College
Biotechnology Workshop
Instructor: Elmar Schmid, Ph.D.
g. Pipette 2 μl from each ligation reaction (1  5) to a sterile 0.5ml PP reaction
tube and place these four tubes on ice; we will use these ligation aliquots in
the following transformation steps (see Section 2.7 below)
 label these four tubes 1  5 correspondingly to their contents
h. Incubate the remainder (= 8 μl) of your initial ligation reactions 1  5 overnight
in the water bath again at 15oC as an eventually needed “back-up” DNA
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