May 07, 2019

Public workspaceTransformation V.4

DOI
dx.doi.org/10.17504/protocols.io.2nwgdfe
  • 1Istanbul Bilgi University
Rengin Yamur Akbiyik
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DOI: dx.doi.org/10.17504/protocols.io.2nwgdfe
Protocol Citation: Rengin Yamur Akbiyik, Sevval Uysalcan 2019. Transformation. protocols.io https://dx.doi.org/10.17504/protocols.io.2nwgdfe
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: May 07, 2019
Last Modified: May 07, 2019
Protocol Integer ID: 22966
Keywords: Transformation
Abstract
Transformation is a direct modification of the genotype of a cell from a different one by extracellular applications using recombinant DNA techniques. Transformation refers generally to the integration of exogenous DNA into the cell and its integration into the genome. Before starting the transformation process, the gene of interest is ligated with the plasmid vector via the ligase enzyme. This process is called ligation. Generally, nucleic acids or plasmids cannot enter into bacterial cells by themselves. The stimulatory effect is required for that purpose. This means that the cell membranes to be transformed must be pre-arranged. Bacteria that can contain free DNA are called competent bacteria. Some bacteria are highly competitive in normal growth conditions; but some must be treated with chemical or physical methods to gain competitive properties. Competent cells which are cells are generally stimulated with calcium chloride about chemically and the cell membranes are arranged such that the plasmid vector containing the gene of interest can harbor the vector.
Some bacterial cells are lasting competent, however; most of them need to be influenced for
being competent. There are two ways about this transformation process that;
• Chemical Transformation
• Electrical Transformation (Electroporation)
In general, chemical transformation is used. In chemical way, some special chemicals are used to open the pores which are found in cell membrane. This is because, opening the pores represents the availability of being permeable. Most of the time, divalent ions like Ca++ are used with assistance of heat-shock.
Materials
STEP MATERIALS
ReagentLB BrothAmrescoCatalog #J106-2KG
Protocol materials
ReagentLB BrothAmrescoCatalog #J106-2KG
ReagentLB BrothAmrescoCatalog #J106-2KG
Add DNA to competent cells
Add DNA to competent cells

Let stand on ice for 30 min
Let stand on ice for 30 min
Duration00:30:00 On ice
Heat shock
Heat shock
Duration00:01:00
Temperature42 °C
Equipment
new equipment
NAME
Grant Bio PHMT Thermoshaker
BRAND
125-0649
SKU
Leave on ice
Leave on ice
Duration00:01:00
Add LB
Add LB
Amount900 µL for 100 µl cells
Amount800 µL for 200 µl cells
ReagentLB BrothAmrescoCatalog #J106-2KG
Equipment
new equipment
NAME
Shaker
BRAND
-
SKU
Put in a shaker
Put in a shaker
Temperature37 °C
Duration00:50:00
Centrifuge at 7000 rpm
Centrifuge at 7000 rpm
Duration00:01:00
Equipment
new equipment
NAME
Centrifuge
BRAND
-
SKU
Discard LB
Discard LB

Resuspend cells and plate them all
Resuspend cells and plate them all