Hi, I'm Marcus to from the laboratory of Steven Helm in the Department of Microbiology and Immunology at the University of British Columbia. Today we'll show you a procedure for the construction of a large insert environmental library. Assisting me in today's procedure is San Lee, who will be performing phage packaging and infection of the library host e coli, as well as Elise Hawley and Chin Yang who will be performing the colony picking.
We have this procedure in our laboratory to study microbio community structure and metabolism in marine and terrestrial environments. So let's get started. This protocol for creating a fo smid DNA library begins following isolation and shearing of genomic DNA from biomass samples procedures that we have detailed in accompanying Jo of articles.
The next few steps prepare the SHE DNA for ligation into the SMID vector PCC one. The first step is to repair the ends of the SHE pieces of DNA and phosphorylate. The five prime ends, all reagents necessary for this step are included in the PCC one SMID library production kit from epicenter to set up the end repair reaction, thaw all reagents on ice, then combine the end repair buffer DNTP, mix a TP up to 20 micrograms of she insert DNA at 0.5 micrograms per microliter enzyme mix and water To reach 80 microliters briefly centrifuge the tube to get all liquid to the bottom.
Incubate the end repair reaction at room temperature for 45 minutes to an hour. Then heat, he inactivate the enzymes at 70 degrees Celsius for 10 minutes. During the end repair incubation, you can prepare the gel for the next step.
Pulsed field gel electrophoresis or PFGE, your goal is to isolate DNA molecules ranging from 30 to 60. Kilobases PFGE is an electrophoretic method for resolving these large fragments which run together during standard electrophoresis. To begin, prepare a 1%gel using low melting aros in 150 milliliters of one XTAE running buffer.
Choose combs that will allow you to load the entire 80 microliter sample of end repair DNA plus loading buffer into one or two wells. Be especially careful handling the gel as low melting agros gels. Break very easily while the gel is solidifying.
Prepare the electrophoresis chamber. Add 2.2 liters of one XTAE into the chamber and and turn on the machine to circulate the running buffer. Set the cooling device to 14 degrees Celsius and set the pump to 70 RPM.
Include a mid-range one PFG marker also from NEB. To avoid cutting your gel too high extrude aros from the gel syringe and slice a small plug from the end with the scalpel. Place the plug at the front of the well and seal with molten aros.
Make sure that the running buffer has cooled to 14 degrees Celsius before you load your gel. In the first well load five microliters of New England Biolabs Lambda Hindi three digest. In the second well load one microliter of the Smid control size marker diluted in 10 microliters of sterile water plus loading buffer.
Then load your end repaired and heat inactivated mix plus loading buffer. Close the lid and program the machine. The gel will run overnight or 12 hours.
When the gel is done running, use a cyber gold stain to visualize the DNA dilute the cyber gold. In 250 milliliters of one XTAE, place your gel into the solution and stain for one hour in the dark cyberg gold is light sensitive While the gel is staining. Prepare a water bath at 70 degrees Celsius and one F 45 degrees Celsius for the gel extraction step that follows.
The next step is to excise and gel. Purify the DNA within your desired size range of 30 to 60 kilobases. Take your stained gel together with an empty teared and labeled micro subterfuge tube and a sterile scalpel to the blue light transluminator.
Line the transluminator with plastic wrap to avoid cross contaminating your gel. Dawn the viewing glasses and turn on the blue light. Now you can see your end repaired genomic DNA and the FO smid control size marker With the scalpel, excise a gel slice five to seven millimeters wide that migrated with and slightly above the smid control size marker.
Use the PFG marker as an upper boundary for your slice. Transfer the slice to the teared tube. You can store the slices at minus 20 degrees Celsius for up to one year before proceeding.
Now it is time to gel. Purify the DNA from your slice. Weigh the tube containing your gel slice and calculate the weight of the slice by subtracting the tear weight.
Next, melt the slice in the 70 degrees Celsius water bath for 10 to 15 minutes. After your gel slice is completely melted, transfer the tube quickly to the 45 degrees Celsius water bath. Keeping in mind that one milligram of solid gel has a volume of one microliter after melting.
Add one unit or one microliter of Jase enzyme preparation per 100 microliters of melted aros. Incubate at 45 degrees Celsius for one hour. Then add two to four microliters more les and incubate at 45 degrees Celsius for one hour more heat.
Inactivate the gel at 70 degrees Celsius for 10 minutes after heat. Inactivating the enzyme, placed the tube on ice for 10 minutes than centrifuge at maximum speed. 13, 000 RPM for 15 minutes to pellet any insoluble particles.
Being careful not to disturb the pellet. Remove the upper supernatant containing your purified DNA and transfer it to a 15 milliliter falcon tube. Then dute it with three milliliters of sterile water next to wash and concentrate the DNA.
Transfer the solution from the 15 milliliter Falcon tube to Ancon Ultra four centrifugal filter unit with an ultra cell 10 membrane. Place the filter unit in a swinging bucket rotor using a 50 milliliter falcon tube as an adapter and spin at 4, 000 GS for six to eight minutes. Centrifuge until approximately 100 to 500 microliters of solution.
Remain on the filter and discard the flow through. Then add another three milliliters of sterile water to the empty falcon tube. Transfer the solution to the Amon tube and repeat the centrifugation step.
Wash the falcon tube a third time and reduce the final volume collected on the filter to 50 to 100 microliters. You do not need to worry about losing the sample. The filter will retain 50 microliters of liquid even after extended centrifugation.
Transfer the remaining DNA solution from the AmCon tube into a pre-washed MicroCon YM 50 centrifugal filter unit and rinse the Amon tube with an additional 50 microliters of sterile water to recover all DNA centrifuge. The MicroCon YM 50 centrifugal filter unit in a micro centrifuge at 10, 000 GS until the filter is still slightly covered with liquid. Check every minute.
If only a small amount of liquid is left on the filter. Turn the filter upside down and recover your DNA solution by a second centrifugation step at 1000 GS for three minutes in a fresh micro centrifuge tube. The resulting volume should not exceed 10 to 15 microliters in total.
Otherwise, your DNA might be too diluted for the ligation. Check the concentration of the resulting DNA solution by NanoDrop or Pico Green Assay. Recovered and repaired.
DNA is now ready to be ligated to the PCC one FO SMID cloning vector for the ligation thaw all reagents on ice. Then combined in the order listed here, mixing after every addition. First, add sterile water to reach a 10 microliter reaction volume.
Then 10 x fast link ligation buffer, 10 millimolar A-T-P-P-C-C one SMID vector and insert at a 10 to one molar ratio. And finally fast link DNA Ligase. Briefly centrifuge the tube to get all the liquid to the bottom.
Tap the tube and spin again. Incubate the ligation reaction at room temperature for two hours and heat inactivate the enzyme for 10 minutes at 70 degrees Celsius. At this point, you can either store the ligation reaction at minus 20 degrees Celsius or proceed to the phage packaging step.
The next step is to generate bacteria phages that will function to introduce your FO SMID library into e coli two or three days before you plan to do the phage packaging step streak out the provided EPI 300 T one resistant plating strain on a plain LB plate and incubate overnight at 37 to degrees Celsius to obtain single colonies. You can seal this plate and store it at four degrees Celsius for future use. The day before the packaging reactions inoculate 50 milliliters of Albee broth plus 10 millimolar magnesium sulfate with a single colony from your Epi 300 plate Shake at 225 RPM and 37 degrees Celsius overnight.
The day of the packaging reactions inoculate fresh 50 milliliter LB broth plus 10 millimolar magnesium sulfate with five milliliters of the overnight culture. Culture, the bacteria at 37 degrees Celsius to an OD 600 of 0.8 to 1.0. Do not exceed OD 600 of 1.0.
Dilute your sample before measuring on the spectrophotometer so that you get an accurate result. Store on ice or at four degrees Celsius until you are ready to proceed or up to 72 hours. Now that the bacteria are ready for infection, it's time to prepare the phage thaw.
The max plaques Lambda Packaging extracts on ice. Using one tube per ligation reaction, thawing will take about 10 to 15 minutes. Meanwhile, pre chill an empty 1.5 milliliter tube on ice again using one tube per reaction.
Once the Lambda packaging extracts have thawed, immediately transfer to 25 microliters of each packaging extract to the pre chilled 1.5 milliliter micro fuge tube and place on ice. Return remaining packaging extract to minus 80 degrees Celsius. Leaving the thawed packaging extracts on ice too long will reduce infection efficiency.
Next, add 10 microliters of the ligation reaction to each 25 microliters of the thawed extracts on ice mixed by pipetting the solution several times without introducing air bubbles. Briefly centrifuge to consolidate all the liquid at the bottom of the tubes and incubate the packaging reactions in a 30 degree Celsius water bath for 90 minutes. After 80 minutes of incubation, thaw remaining packaging extract on ice.
After the 90 minute packaging reaction is complete, add the remaining 25 microliters of packaging extract to each reaction tube. Incubate reactions for an additional 90 minutes at 30 degrees celsius. After the second incubation, add 100 microliters of the prepared phage dilution buffer in each packaging tube and mix gently add five microliters of chloroform to each tube.
Now you should have a total volume of 165 microliters in the tube. A viscous precipitate may form after chloroform edition. Avoid this precipitate as well as the organic chloroform phase when pipetting the phage particles.
At this point, you can store the packaged phage particles at four degrees for several days if necessary, but use freshly prepared phage for best results. Now you are ready to infect the host cells, which you have been growing up throughout the phage Packaging steps with the packaged phage. Add packaged phage to EPI 300 T one resistant host cells OD 600.8 to 1.0 in the ratio of 400 microliters of epi 300 T one resistant cells for every 10 microliters of phage particles.
To avoid pipetting any chloroform from the bottom of the tube of packaged phage, we use 125 microliters of the packaged phage particles and five milliliters of prepared epi 300 T one resistant host cells mixed gently and then incubate at 37 degrees Celsius for 30 minutes. The next step is to determine the titer of the packaged phage. You do this by plating the infected bacteria on medium to select infected cells.
Then counting the resulting colonies and dividing by the volume of phage added to the bacteria you plated. The C one SMID contains a chloramphenicol resistance gene. So to select for infected clones plate the bacteria out on chloramphenicol containing LB plates.
Spread two times 50 microliters and two times 10 microliters of the phage infected EPI 300 T one resistant cells on four separate plates and place the plates upside down in a 37 degrees Celsius incubator for 16 to 24 hours, no more than 48 hours until colonies form. Meanwhile, harvest the residual EPI 300 T one resistant cells from the five milliliter infection by centrifugation at 3, 500 GS for 10 minutes at four degrees Celsius. Discard the supernatant.
Add one milliliter of LB with 20%glycerol to the tube resus. Suspend the cell pellet and quat to 100 microliters each in two milliliter cryo vial tubes freeze immediately and store at minus 80 degrees Celsius. For further use in the colony picking step the next day, count the number of clones on the four plates in the 37 degrees Celsius incubator and determine the titer of the packaged phage, which you use in the next step.
The frozen EPI 300 T one resistant glycerol stock contains cells transduced with all of the different plasmids in your library. Next, spread an appropriate amount based on the viral titer and plate size of each smid. Clone on 2 45 by 2 45 millimeter square.
Bioassay plates of LB with chloramphenicol using glass beads. Also add a small amount of LB broth to the plate to spread your cells evenly. Keep in mind that preparing the glycerol stock additionally concentrates your cells about fivefold.
Incubate at 37 degrees Celsius for 24 hours on the days of colony picking, prepare 96 or 3 84 well plates filled with 200 microliters or 70 microliters per well of LB broth. Supplemented with 12.5 micrograms chloramphenicol per milliliter of broth for antibiotic selection and 10%glycerol for freezing. We use the automated qfi three plate filling system for this step.
The next step is to pick colonies from your prepared LB CHLORAMPHENICOL plates into your prepared 96 or 3 84 well plates for freezing. For this step, we are using the clone picking robot qix two, according to the user's manual, to create a sterile environment around the clone picking area. Turn on the UV light for 30 minutes while the UV light is on.
Prepare 500 milliliters of 1%bleach, 500 milliliters of sterile deionized water and 500 milliliters of 80%ethanol. When the UV light shuts off, fill up the trays as labeled from back to front. Fill in 1%bleach, then autoclave deionized water, and 80%ethanol in the front tray.
Make sure they are full as picking needles cannot be washed properly without sufficient washing solution. After setting up the parameters for optimal picking, take the lids off your bacterial plates and your LB filled 96 or 3 84 well plates and position them between the posts in the Q picks. Working area plates should be tight against front right corner so they cannot move during picking.
Then close the front panel. Once all parameters are set, the picking procedure can begin. When all picking is completed, rinse the brushes and trays with deionized water and set on the bench to dry.
Clean up any spills that may have occurred and wipe down the inside of the CICS with 80%ethanol. Ensure that all glycerol stock plates are labeled and place them in the 37 degrees Celsius incubator for 24 hours to grow a culture from each picked colony. Leave a note on the incubator that contains the date number of plates incubated, time of incubation, time for removal, and your initials.
After the incubation, you have your library. Each well contains a culture of one bacterial clone containing the PCC one fo smid ligated to one piece of sheer DNA from your starting sample. Freeze the plates at minus 80 degrees Celsius for long-term storage and congratulate yourself on a job well done.
The presented protocol describes the procedure of how to generate an environmental smid library to capture the genetic content of a microbial community in a given habitat. This protocol should create a SMID library, which is representative to the genetic content of the sampled environment, and should enable the reader to optimize critical steps. If the amount of FO smid clones obtained at the end of the whole procedure is too low.
We've just shown you how to construct the FO SMID library from genomic DNA derived from a marine water sample. When doing this procedure, it is important to start with genomic DNA of high quality. Have all your plates and reagents ready before starting and plan enough time for this multi step process.
So that's it. Thanks for watching and good luck with your experiments.
