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작성자마황 조회 15회 작성일 2020-10-17 07:58:30 댓글 0

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Competent Cell Transformation

Visit http://www.invitrogen.com/compcells for more information.

Overview of chemical transformation

This video will walk you through the basics of chemical transformation. Transformation is the process by which bacterial cells take up foreign DNA from their environment. Typically, this is done in the lab for two main reasons: to propagate a recombinant plasmid or to obtain the results of a sub-cloning reaction.

There are two main classes of competent cells: chemical and electro-competent. The procedure shown in this video can be used with most chemically competent cells. We are focusing on the protocol provided with Invitrogen's Top 10 strain of comp cell.

Getting Started

First, let's talk about storage conditions and what comes with your kit. Competent cells are stored at minus 80° degrees. Invitrogen's kits come with vials of competent cells, transformation instructions, a vial of S.O.C. medium, and a pUC19 transformation control.

The OneShot® Top 10 kit used in this video comes with vials containing 50 micro liters of competent cells; enough for one transformation per tube. The competent cells must remain frozen until just before you are ready to use them. If they thaw too soon, it can affect the transformation efficiency.

For your transformation protocol, you will need the following items:

- A water bath set at 42° degrees
- An ice bucket with ice
- A 37° degree shaking incubator
- A 37° degree incubator
- 10 cm diameter LB Agar plates with the appropriate antibiotic

In this case a 100 micrograms per microliter of ampicillin

The selective plates should be warmed in an incubator for 30 minutes before you use them. You will need one plate for each transformation. Finally, you will need the vial of S.O.C. medium thawed and at room temperature.

The first step is to mix the comp cells and the plasmid DNA of interest. Briefly centrifuge the DNA and put it on ice. This DNA can be from a ligation reaction or plasmid DNA you wish to propagate. Next, thaw on ice one 50 microliter OneShot™ vial of cells for each transformation reaction.

Pipette one to five microliters of your DNA samples directly into each vial of competent cells. Mix by tapping gently and not by pipetting up and down. Your remaining ligation reaction can be stored at minus 20° degrees. We also highly recommend setting up a transformation control with the PUC 19 provided to ensure the transformation was performed correctly and the competent cells were at the expected competency.

Add one microliter of the pUC19 control plasmid to 50 microliters of competent cells and tap gently to mix. The next step is to incubate the cells on ice for 30 minutes. Next is the heat shock step. This is harsh on the cells, so be sure not to mix or shake the vials. Incubate for exactly 30 seconds in the 42° degree water bath. After the incubation, remove and place on ice.

Now the cells are allowed to recover from the transformation and are grown in rich S.O.C. medium. Add 250 microliters of the pre-warmed S.O.C. medium to each vial. Next, place the vials in a microcentrifuge rack inside the shaking incubator. Or, tape the vials on their sides to the shaking platform in the incubator. Shake the vials at 37°C for one hour at 225 rpm. If you do not have a shaking incubator you can add the vials to a 37°C regular incubator.

Once the cells have recovered, it is time to plate them. The antibiotic you added will select only transformed cells. Pipette 15 microliters from the individual vial onto its own labeled LB plate. The remaining transformation mix may be stored at 4° and plated the next day if desired. For the control, remove 10 microliters from the vial and mix with 20 microliters of fresh S.O.C. and plate the entire 30 microliters as you did with the other transformations.

To spread the cells evenly across the plate add 5 to 8 sterile glass beads and rotate, or use a sterile glass rod to spread across the plate. After removing the glass beads, invert the plates and incubate in 37° degrees overnight. Plasmid containing the antibiotic resistance marker that allowed them to grow in the presence of a selection antibiotic; the pUC19 control plate will have a larger number of colonies than your ligation plates.

Many people also use blue white screening for selecting their clones. Each of these is a transformant and has taken up the plasmid containing the antibiotic resistance marker that allowed them to grow in the presence of the selection antibiotic. The pUC19 control plate will have a larger number of colonies than your ligation plates. Many people also use blue white screening for selecting their clones.

For more trouble shooting tips or information on all competent cell structures visit http://www.invitrogen.com/compcells
Slimane Dz : Isnt 15 microliter too little to plate on selective LB?
Alan Wedgeworth : can you get a charge from a dead computer batory
Abhinadh Varun : Nice but . Wat u dng.
Ananda Ayyappan JV : Self made chemocompetent cells---5min on ice with DNA-Heat shock-5min ice-45min shaking at 37deg--plating as desired!
(works best for me since 3 years!)
Nathan Ortiz : Long story short bioshock
Bernardo Alabés : i want to work for invitrogen

Plato Шлёпанцы модель JM109



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SZERETET, FÉNY Mindenkinek.

... 

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