Oct 14, 2020
Restriction enzyme & gel electrophoresis
This protocol is a draft, published without a DOI.
- 1Chung Shan Medical University
Protocol Citation: Hung Liang Pai 2020. Restriction enzyme & gel electrophoresis. protocols.io https://protocols.io/view/restriction-enzyme-amp-gel-electrophoresis-bh67j9hn
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: Working
We use this protocol and it's working
Created: July 02, 2020
Last Modified: October 14, 2020
Protocol Integer ID: 38847
Preparation
Preparation
Set the dry bath incubator,37 °C
Take 10x buffer from the -20 °C fridge to thaw.
Protocol-Restriction enzyme
Protocol-Restriction enzyme
To add 500 ng DNA for each sample into each eppendorf, calculate the required DNA volume for each sample. The volume is decided by the formula below:
Volume(sample)=500 ng / concentration measured by spectrophotometer(ng/uL)
Calculate the required ddH2O volume for each sample. The volume is decided by the formula below:
Volume(ddH2O)=20uL - 2uL - 0.5uL - 0.5uL - Volume(sample) uL
Take sufficient eppendorfs and mark them for each sample. First, add ddH2O based on previous calculation in each eppendorf. Next, add DNA samples based on previous calculation in each eppendorf.
Take an eppendorf and mark as CT(cocktail). Add 10x buffer to the eppendorf. The volume is decided by the formula below:
Volume(10x buffer)=2uL x (the number of samples +1)
Take the determined restriction enzyme from the-20 °C fridge . Add RE to the previous eppendorf respectively. Remember to put restriction enzyme On ice during the whole process and take RE back to the-20 °C fridge right after the process ASAP. The volume is decided by the formula below:
Volume(RE 1)=0.5uL x (the number of samples +1) Volume(RE 2)=0.5uL x (the number of samples +1)
Add3 µL of CT in each eppendorf. Vortex and spin down each eppendorf to make the liquid stay at the bottom.
Put each eppendorf in the 37 °C dry bath incubator for 02:00:00 or even more.
Protocol-Gel electrophoresis
Protocol-Gel electrophoresis
Put a gel into the electrophoresis tank.
Take a duran youtility. Add 3.5 mL TAE and 350 mL ddH2O into it and then mix it. Put it into the electrophoresis tank. Make sure the gel is soaked into the liquid completely.
Take 5 µL of marker (1kb) and load it in the first well.
Take loading dye and drop on the parafilm separately. The volume is decided by the formula below:
Volume(loading dye)=1uL x (the number of samples)
Take5 µL of sample reacted with RE for 02:00:00 . After pipetting with 1 µL loading dye, add it into each well. Repeat this step until all the samples are filled into each well.
Connect wires with the power supply. Set time for 00:30:00 and click "start" button. Make sure there are bubbles appearing next to anode and cathode.
After gel electrophoresis, put the gel into gel reading machine and report the result.