Transformation of competent cells and clone screening Written by Steve Doyle (s.doyle@latrobe.edu.au) - March 2014 - - - transformations involve taking intact plasmid DNA, or ligated plasmid/PCR product, and using bacteria to cultivate them bacteria take up some of the DNA during the heat-shock process at 42C, when small pores form in the bacterial membrane bacteria that do take up a plasmid gain a selective advantage; plasmids contain antibiotic resistance genes in them, and when spread onto on an agar plate that contains antibiotics, only those bacteria that contain a plasmid will survive this results in small colonies of bacteria to form on the plate; each colony contains many identical bacteria that all derive from a single bacterium that took up the DNA each colony is considered an independent clone, and within each colony, the DNA can be considered identical, but between colonies, each is considered potentially different; we therefore usually screen a few different colonies from each plate to find one that we want this protocol demonstrates how to do the transformations, how to screen colonies for the plasmid and insert (colony PCR), and how to preserve the bacteria containing the plasmid DNA for future use (glycerol stocks) Preparation - LB broth - LB / agar plates containing appropriate antibiotic - 30% glycerol / LB - competent cells Protocol Day 1 1. add 2 ul of ligation mix / plasmid DNA (1-10 ng) to a sterile, chilled microcentrifuge tube 2. transfer 100 ul of competent cells to the tube and swirl with the pipette tip to mix with the DNA a. do not pipette up and down to mix – this will negatively affect that transformation 3. keep tube on ice for 30 mins 4. place tube in a preheated heat block / water bath set to 42°C for exactly 90 seconds a. be gentle on do not shake tubes 5. quickly but gentle transfer tubes into ice and allow cells to chill for 1-2 minutes 6. add 500 ul of LB to each tube 7. incubate tubes in a 37°C shaking incubator for 45 minutes a. this allows the cells to recover and express the antibiotic resistance gene 8. once removed from the incubator, transfer up to 200 ul onto an agar plate containing the appropriate antibiotics a. ampicillin for pGem-T-Easy cloning 9. spread cells using a sterilized bent glass rod a. to sterilize, dip the rod into ethanol and then quickly pass it through the flame of a Bunsen burner. Do not hold in the flame, but let it burn out by itself. Allow a could of seconds to cool before spreading. 10. Invert the plates, make sure the plate has been labeled appropriately, and incubate at 37°C for approximately 16 hours a. Do not leave it for too long in the incubator, as non-specific satellite colonies will appear Day 2 11. Check the plates first thing in the morning. Hopefully they should have some discrete colonies (white raised spots) growing on the plates. You want the colonies to be between 1-2 mm wide – no more. If they are too small, put back in the incubators for another hour or two. When they are ready, take them out of the incubator and place on the bench. 12. Proceed with either a. Colony PCR preparation; OR b. Making overnight cultures Colony PCR preparation 1. determine how many colonies you want to screen from each plate a. if you expect there to be some diversity among clones on the plate, (ie. you are confirming a heterozygote SNP, or you know you have mixed DNA sequences) pick >5 b. if you expect the colonies to be essentially the same, pick 3 2. setup one sterile microcentrifuge tube per colony to be screened and into it, aliquot 20 ul of H2O 3. using a P20 pipette and pipette tip, gently touch the end of the tip onto a colony and transfer it into one of the tubes, swirling and pipetting up and down to mix a. you do not need much of the colony to be transferred for this to work; importantly, you do not want more than one colony being transferred. Avoid colonies that are too close together. 4. Repeat for all colonies to be screened 5. Set up single tubes, 8-well strips or 96-well plates (depending on how many colonies you have to screen) 6. Add 10 ul of each colony resuspension to individual wells, keeping the remaining 10 ul aside on the bench – DO NOT DISCARD. 7. Place the tube/strip/plate in the PCR machine and incubate a 95°C for 20 minutes to lyse the bacterial cells a. This becomes the template for the PCR 8. Proceed with a relevant PCR amplification, using 1 ul of the bacterial lysate as the template DNA for the reaction a. Primers could be insert specific, plasmid backbone specific (M13, T7), or a combination of both 9. Once you have confirmed which colonies are positive by PCR and running a gel, use the remaining 10 ul of bacterial suspension (NOT the lysate) as a starter overnight culture – proceed with Making Overnight Cultures Making Overnight Cultures 1. prepare for making overnight cultures but determining how many colonies you are going screen 2. per colony, aliquot 5 ml of LB broth into a 10 ml tube 3. add sufficient antibiotic to each tube a. if using pGem-T-Easy, add 5 ul of 100 ug/ml of ampicillin to each tube 4. using a P20 pipette and pipette tip, gently touch the end of the tip onto a colony and transfer it into one of the tubes, swirling and pipetting up and down to mix a. you do not need much of the colony to be transferred for this to work; importantly, you do not want more than one colony being transferred. Avoid colonies that are too close together. b. If you are proceeding from the colony PCR step, add the entire remaining 10 ul of bacterial suspension to the tube 5. Place tubes on roughly a 45 degree angle in an 37C shaking incubator (180 RPM) for overnight a. Placing the tubes on an angle helps aerate the suspension, promoting bacterial growth. Tubes straight up or lying down do not grow as well. Day 3 6. In the morning, check the tubes – they should have changed from clear yesterday to be cloudy today, which means that the bacteria have successfully grown 7. For samples that have grown, prepare a glycerol stock of the sample before proceeding with the miniprep DNA extraction a. Glycerol stocks of bacterial cultures can be kept essentially indefinitely in the -80C freezer. You only really need to make them if you plan to continue to use the DNA you extract time and time again. 8. Preparing glycerol stocks a. Per sample, add 500 ul of 30% glycerol/LB into a microcentrifuge tube i. 30% glycerol/LB = 30 ml glycerol + 70 ml LB broth; autoclave to sterilise 9. b. Add 500 ul of the bacterial suspension to the 30% glycerol / LB c. Place in the -80C freezer Once glycerol stocks are made, proceed to extract the plasmid DNA using a Miniprep DNA extraction kit, following the manufacturers instructions a. We typically use the entire remaining ~4.5 mls in the Miniprep 10. After you have done your Miniprep, and confirmed your insert is correct, ie. by Sanger Sequencing, you do not need to keep ALL of the glycerol stocks that you made in step 8 above – just keep the one that has been confirmed.