Competent Cell 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 procedures shown in this video can be used with most chemically competent cells we are focusing on the protocol provided with in vitro jhin's top 10 strain of calm cell first let's talk about storage conditions and what comes with your kit competent cells are stored at minus 80 degrees in vitro jhin's kits come with vials of competent cells transformation instructions a vial of soc medium and a puck 19 transformation control the one shot top 10 kit used in this video comes with vials containing 50 microliters 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 thought 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 and a 37 degree incubator 10 centimeters diameter lb agar plates with the appropriate antibiotic in this case a 100 micrograms per milliliter 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 SOC 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 150 micro litre one shot 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 PUC 19 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 vial 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 SOC medium add 250 microliters of the pre-warmed SOC medium to each vial next place the vials in a micro centrifuge rack inside the shaking incubator or tape the vials on their sides to the shaking platform in the incubator shake the vials at 37 degrees Celsius for one hour at 225 rpm if you do not have a shaking incubator you can add the vials to a 37 degrees Celsius 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 degrees and plated the next day if desired for the control remove 10 microliters from the vial and mix with 20 microliters of fresh SOC and plate the entire 30 microliters as you did with the other transformations to spread the cells evenly across the plate add five to eight sterile glass beads and rotate or use a sterile glass rug to spread across the plate after removing the glass beads invert the plates and incubate in 37 degrees overnight ESMA containing the antibiotic resistance marker that allowed them to grow in the presence of the selection antibiotic the puck 19 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 transforming and has taken up the plasmid containing the antibiotic resistance marker that allowed them to grow in the presence of the selection antibiotic the puck 19 control plate will have a larger number of colonies than your ligation plates many people also use blue white screening for selecting their clones so what happens if you go to the incubator and your transformation did not work and you have no colonies transformation works almost every time but occasionally it doesn't here are some areas to check ensure the antibiotic in the agar plates matches the clone resistance marker and is at the correct concentration impurities in your DNA sample can also affect transformation ensure phenol proteins ethanol and detergents have been removed from the DNA ensure there is not an excess of DNA or volume also ensure the competent cells have been handled correctly use immediately after thaw and do not vortex ensure the incubators and water baths are at the correct temperature setting for more troubleshooting tips or information on all competent cell strains visit WWN beat region comm slash comp cells

Channel: Thermo Fisher Scientific

Views: 93,260

Rating: 4.9214144 out of 5

Keywords: competent cell, chemical transformation, bacterial cells, recombinant plasmid, sub-cloning reaction, chemically competent cells, S.O.C. medium, pUC19, cloning, bacterial transformation, OneShot, blue white screening, antibiotic resistance marker

Id: 8admZaGsFHo

Length: 6min 58sec (418 seconds)

Published: Thu Sep 15 2011