Add 2.15 g of MS basal salt mixture, 10 g sucrose to a flask containing ~0.8 L of distilled water and stir to dissolve. Check and adjust pH to 5.6-5.8 using 1 M potassium hydroxide (KOH). Add distilled water to make 1 L.
Divide the media into two 1 L flasks, 500 mL each. Add 4g of agar to each flask. Add a magnetic stir bar to each flask. Cover with foil.
Autoclave for 45 min.
After autoclaving, place the bottles in a water-bath heated to 60-65°C, and allow the MS medium to cool (around when the bottle can be held with bare hands).
Starting from this step, perform all the steps in sterile conditions in a laminar flow hood.
If adding BASTA: Add 500 µL BASTA (50mg/ml) to the bottle and stir the MS medium to evenly distribute.
Pour enough media into plates to cover approximately half of the depth of the plate.
Allow the plates to cool at room temperature for about 1 h to allow the agar to solidify.
Note: If the plates are not to be used immediately, carefully place them in a plastic bag agar-side down and store at 4 °C.
Adapted from:
Lindsey BE, Rivero L, Calhoun CS, Grotewold E, Brkljacic J. Standardized Method for High-throughput Sterilization of Arabidopsis Seeds. Journal of Visualized Experiments : JoVE. 2017;(128):56587. doi:10.3791/56587.
Prepare 50% (v/v) bleach solution to be used for sterilizing the seeds. To dilute bleach, add 100 mL of bleach to 100 mL of distilled water. Add 50 µL of Tween 20 detergent to the bleach solution. Note: Prepared bleach solution can be stored for up to a month as long as it is only opened in sterile conditions.
Aliquot 100 seeds into a 1.5 mL microcentrifuge tube.
Sterilize the seeds using a 50% bleach solution. Add ~500 µL of the 50% bleach solution to the microcentrifuge tube containing the seeds. Vortex briefly or tap the bottom of the tube to suspend the seeds in the bleach solution. Place the tubes on the Nutator to keep the solution agitated.
The remainder of the protocol needs to be performed in a laminar flow hood.
After 10 min, remove the bleach solution from the microcentrifuge tube using a pipette or an aspirator fitted with a pipette tip on the end.
Add ~500 µL of sterile distilled water to the tube. Close the tube and invert to mix. Allow seeds to settle to the bottom of the tube. Once seeds have settled to the bottom of the tube carefully remove the bleach solution by pipetting. Repeat this rinsing process 6 times.
Add 1 mL of autoclaved 0.15% (w/v) agar to the tube to suspend the seeds.
In a laminar flow hood, label the bottom of the MS plate with the stock name and the current date.
Spread the seeds around the MS plate using a sterile, single-use inoculating loop or a sterile pipette tip or a sterile, single-use Pasteur pipette. Note: If seeds are to be sown in rows, a pipette with a 200 µL tip can be used to individually place seeds in the desired positions. To improve the flow of seeds, the end of the pipette tip can be trimmed by 3-5 mm using scissors. Any misplaced seeds or clusters of seeds can then be repositioned or separated using a sterile single-use inoculating loop.
Place the lid on the MS plate. Seal the MS plate by wrapping the plate with microporous tape.
Place the plates with the lid on top for three days at 4 ˚C and ambient humidity. Note: This process is called stratification and serves to synchronize the germination of individual seeds.
Transfer the plates to the growth environment. Maintain temperature at 23 °C and light intensity at 120 – 150 µmol/m2s with 16 h light/ 8 h dark photoperiod. Place plates flat into the grow environment with the lid on the top so the roots grow into the medium.
Let the seedlings on plates grow for 8 days. Note: An 8 day growing period allows late germinating seeds to germinate. Plates can be scored sooner than 8 days if all seeds have germinated.
Adapted from:
Lindsey BE, Rivero L, Calhoun CS, Grotewold E, Brkljacic J. Standardized Method for High-throughput Sterilization of Arabidopsis Seeds. Journal of Visualized Experiments : JoVE. 2017;(128):56587. doi:10.3791/56587.
1) I use Q5 for colony PCR, and have not tried it very often with other polymerases.
2) For my typical workflow I find it more efficient to make ‘blind’ liquid cultures from colonies and then screen the liquid, rather than inoculate liquid cultures and screen colonies from a plate at the same time. This protocol reflects that but can be easily tweaked to use plate colonies.
3) Following from (2), for one or more positive cultures I typically want to make a glycerol stock and then purify plasmid from the remainder of the tube. So, I open all the tubes to be screened in the hood and aliquot into strip tubes to allow quick loading of the PCR with a multipipettor. I take the strip out of the hood to my bench to set up the PCR, and leave the tubes in the hood while waiting for the PCR results so that I can again aliquot culture for glycerol stocks from the positive cultures under sterile conditions. Your workflow might vary; I also worry to a likely unnecessary degree about contamination of bacterial cultures from the environment.
4) I use a 30 uL PCR reaction volume to reduce the risk of inhibition from the culture; if you try and it doesn’t work, you can increase the volume to 50 uL while leaving the volume of bacteria the same.
5) Spray a test tube rack with ethanol, put your test tubes of culture in and spray all around the outside of the tubes with ethanol. Lightly spray a 200 uL pipettor with ethanol as well, and if there are not already 20/200 uL tips in the hood, spray the outside of an unopened box of tips. Place everything in the hood and let the outside of all the containers dry for ten minutes.
6) While tubes are drying, set up the PCR. I use the following recipe for a first try:
Q5 2x master mix 15 uL
F primer 1.5 uL multiply first 4 ingredients by number of
R primer 1.5 uL samples, then add 10% to make the master mix
water 10 uL
template 12-24 hour E. coli liquid culture 2 uL (can be increased to 3 if cultures are thin)
Add all ingredients to a 1.7 mL microcentrifuge tube, vortex for 3 seconds, spin down in a small centrifuge for 5 seconds, and keep in a cold block until you are ready to move on with the next steps
7) In the hood, aliquot 20 uL of each liquid culture into a labeled strip of tubes. Cap the culture test tubes and leave them in the hood, and take the strip out to the bench.
8) Aliquot 28 uL of master mix into a new strip of labeled tubes (or plate, or multiple strips), set in one of the small green PCR tube cold blocks from freezer #2. You do not need to change tips between wells because all of the wells contain the same thing at this point.
9) Set a 10 or 20 uL multipipettor to 2uL, and pipette 2 uL of liquid culture from the strips you previously aliquoted into the corresponding PCR reaction tubes. Without changing tips and being careful not to splash any drops or bubbles into the wrong tubes, reset the pipettor to the max volume, either 10 or 20 uL. Gently pipette up and down 10 times to mix the reaction, making sure each tip is back in the same well it was originally used for. Do not fully eject the liquid until the final mixing step, otherwise you will likely end up with some liquid stuck in the pipettor. Repeat for any additional strips or wells, changing tips between each strip.
10) Cap the tubes or seal the plate, and leave on the cold block while you set the PCR machine.
11) The initial denaturation step must be lengthened to lyse the bacterial cells in addition to denaturing the DNA. I also use 40 cycles instead of my typical 30 or 35 to account for inhibition by the culture and unpredictable amounts of template I use the following settings:
1) 98 degrees 10 min (preheating step)
2) 98 degrees 10 minutes 20 seconds
3) 98 degrees 15 seconds repeat steps 3-5
4) Tm (calculated with NEB Q5 calculator) 15 seconds 39 times
5) 72 degrees 25 sec/kb 5 sec (e.g. a 200 bp fragment is .2kb*25=5, 5=10sec)
6) 72 degrees 2 minutes
set the hot top to 105 degrees and hit start. It take about 5 minutes for the machine to fully heat up.
12) Once the PCR machine is ready, spin down the PCR tubes in a small centrifuge for 2 seconds. Put the tubes in the machine, then manually skip the first (preheating) step to start the actual cycle. I set the preheating step to 10 minutes rather than infinity as a safeguard to avoid ruining the PCR if I do walk away and forget to advance the program.
13) Once the PCR is done, run on a gel or qiaxcel.
14) For positive cultures identified by the screen, go back to the hood and aliquot 500 uL of culture into a sterile cryovial, then add 500 uL of sterile 50% glycerol. Make sure not to contaminate the bottle of glycerol!
15) Mix the cryovials by inverting several times (do not vortex), then freeze at -80.
16) Use the remaining culture for plasmid extraction (E. coli) or plant transformation (Agrobacterium)
Protocol developed by Eli Hornstein