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Ligation of Opd Insert Into pUC19 Vector: Reagent

Ligation is the process of joining DNA fragments together through enzymatic action. This experiment used T4 DNA ligase to join the opd insert and pUC19 vector fragments, which were previously digested with BamHI. The ligation reaction was incubated at 22°C for 30 minutes, then analyzed by gel electrophoresis. The results showed bright bands for each sample, though one sample had some streaking, possibly due to contamination or primer degradation during the ligation. While most samples showed successful ligation, one had lower concentration of the ligated plasmid.

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Andrea Sanchez
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136 views2 pages

Ligation of Opd Insert Into pUC19 Vector: Reagent

Ligation is the process of joining DNA fragments together through enzymatic action. This experiment used T4 DNA ligase to join the opd insert and pUC19 vector fragments, which were previously digested with BamHI. The ligation reaction was incubated at 22°C for 30 minutes, then analyzed by gel electrophoresis. The results showed bright bands for each sample, though one sample had some streaking, possibly due to contamination or primer degradation during the ligation. While most samples showed successful ligation, one had lower concentration of the ligated plasmid.

Uploaded by

Andrea Sanchez
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Ligation of opd Insert into pUC19 Vector

Andrea Sanchez
October 13, 2020

Ligation of opd Insert into pUC19 Vector

Introduction:

Ligation is the process that involves the joining of linear DNA fragments together with covalent
bond through enzymatic action. For this experiment, utilizing T4 Ligase (T4 bacteriophage) to
join opd and pUC19 fragments through cohesive overhanging ends/blunt ends that results in
phosphodiester bonds between 3’ hydroxy end and 5’ phosphate end. BamHI from the previous
experiment was used to digest opd and pUC19 and create the sticky end fragments for both. T4
will join the overhanging ends and is an important factor for recombinant DNA.

Materials and Methods:

Purified plasmid (opd insert) (session 5)


Purified Vector (pUC 19) (session 5)
T4 Ligase
5x Ligase Buffer
Ice bucket & Ice
Autoclaved MilliQ H2O
Thermal cycler

Materials need to be on ice until they are ready for incubation. Once ready, label your 200μL
tube and combine the following:

Reagent Volume
Purified Insert (opd) 15μL
Purified Vector (pUC 19) 5μL
5x Ligase Buffer 6μL 6μL
MilliQ H2O 2.5μL
T4 Ligase 1.5μL
Total volume 30uL

The Lab Instructor will add T4 Ligase and follow with a vortex for 1-2 seconds, quick spin for 2-
3 seconds, then placed back on the ice. Once samples are ready, the Lab Instructor will place
samples in thermal cycler for 30 minutes at 22 °C. After thermal cycling is complete, return
ligation sample to the ice. Prepare samples for gel QC as follows:

1 KB Ladder Ligation
Reaction
Sample 4 μL 8 μL
4x SDS Dye 4 μL 4 μL
MilliQ H2O 4 μL
Ligation of opd Insert into pUC19 Vector
Andrea Sanchez
October 13, 2020
Afterwards, placed the samples to be incubated at 70 °C for 5 minutes followed by ice bath for 2
minutes. Once done, loaded the samples onto the gel to run at 110 V for approximately 1 hour or
just until the dye front is ¾ distance of the gel.

Results and Discussion:

*For gel electrophoresis, 1X TAE buffer was used, and the gel was run at 110 volts for an hour.

Lane 1 - kb ladder


Lane 2 – Empty
Lane 3 - Group A's ligation result
Lane 4 – Empty
Lane 5 - Group B's ligation result
Lane 6 – Empty
Lane 7 - Group C's ligation result

Taking from our previous lab where it was hard to see the bands from the purified vector and
insert, the sample has now produced very bright bands in comparison. For Group B’s ligation
result there is a streak behind it. This could have resulted from contamination or from primer
degradation. Primers are single stranded and can degrade easily and can result in streaking.

Conclusion:

With visibility now available for our bands but streaks as well, there is a high chance that there
was either contamination or primer degradation. Group B has less concentration when compared
to group A but more compared to group C ligation result.

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