Oct 18, 2021

Public workspaceLigation and gel electrophoresis

DOI
dx.doi.org/10.17504/protocols.io.by5dpy26
  • 1Chung Shan Medical University
  • CSMU_Taiwan
CSMUTaiwan
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DOI: dx.doi.org/10.17504/protocols.io.by5dpy26
Protocol Citation: Chia-Hsien Shih 2021. Ligation and gel electrophoresis. protocols.io https://dx.doi.org/10.17504/protocols.io.by5dpy26
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: October 17, 2021
Last Modified: October 18, 2021
Protocol Integer ID: 54149
Abstract
This protocol is used for ligation of circular probe
Preparation
Preparation
Dilute linear DNA into 10µM

Dilute T4 ligation primer into 10µM
Protocol of Ligation
Protocol of Ligation
Add Amount11 µL of RNase-free water into a eppendorf

Add Amount3 µL of 10X T4 Ligase buffer

Add Amount9 µL of 10µM T4 ligation primer

Add Amount3 µL of 10µM linear DNA

Add Amount4 µL of T4 Ligase

Spin down after vortex
Incubate for Duration04:30:00 at TemperatureRoom temperature

4h 30m
Protocol of Gel Electrophoresis
Protocol of Gel Electrophoresis
30m
30m
Put 2% agarose gel into the electrophoresis tank.
Prepare 0.5X TAE buffer ( add Amount3.5 mL 50X TAE buffer and Amount350 mL ddH2O and mix them), then pour into the electrophoresis tank. Make sure the gel is soaked into the buffer completely.

Take Amount5 µL of marker and load it in the first well.

Take Amount1 µL of 6X loading dye and drop on the parafilm separately, pipetting dye with Amount5 µL of sample

Load samples into each well
Run gel at a constant voltage of 100V forDuration00:30:00 at room
temperature
30m
After gel electrophoresis, put the gel into gel reading machine and report the result.