TITLE: Optical mapping preps for Petunia spp.
AUTHORS: Elena Hilario

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Optical mapping technologies assist the assembly of large and complex genomes. Although protocols for preparing this kind of samples are now conveniently available as kits, some plants will not respond to standardized conditions or the researcher might not have access to kits.</div><div class = "text-block">This protocol describes how to isolate plant nuclei from fresh very young leaves. The nuclei are purified over two Percoll® gradients and embedded in agarose. The nuclear DNA is released by solubizing the nuclear membrane with detergents and removing the proteins by digestion with Proteinse K. The nuclear DNA is analyzed by pulse field gel electrophoresis. If most of the DNA is > 1 Mbp, the sample could be further evaluated for optical mapping analyzis by the service provider.</div></div>

[STEPS]
?. [Tissue Fixation]
Remove ~   and place them in a 50 mL Falcon tube containing ~. Attach the green screened cap and slowly add more Fixing solution (total volume 60 mL).Remove all the air bubbles by tapping the tube but keeping it upright. Close the tube with its own cap and incubate on ice.
Do the tissue fixation and nuclei isolation steps in a fume hood
[very young leaves]
[Fixing solution]
[Formaldehyde fixing reaction]
[Formaldehyde fixing reaction]
?. [Tissue Fixation]
Discard the Fixing solution by pouring it through the green screened cap.
Dispose the formaldehyde waste according to your institution's waste management guidelines.
?. [Tissue Fixation]
Add  through the green screened cap, and remove the air bubbles as in step 1. Incubate on ice.
[0.125 M glycine solution]
[Fixing quenching step]
[Fixing quenching step]
?. [Tissue Fixation]
Discard the glycine solution through the green screened cap. Add  and remove air bubbles as in step 1. Incubate on ice. Repeat this step two more times.
[ice cold 1X PBS solution]
[1X PBS wash]
[1X PBS wash]
?. [Nuclei isolation]
After decanting the last 1X PBS wash, transfer the leaf tissue to the Petri dish containing ~ . Place the Petri dish securely on ice. Finely chop the sample using two scalpel blades, as if they were knife and fork until the pieces are ~ 1 mm.
[ice cold NEB complete]
?. [Nuclei isolation]
Transfer the sample including the buffer to the tissue homogenizer using a spatula and a disposable Pasteur pipette. Pour 5-10 mL of ice cold NEB complete into the homogenizer, insert the pestle and place it on the ice bucket so it is covered by ice up to half way of its length.Homogenize the sample by genlty grinding with the pestle in a spiral movement, and lift it just below the liquid level. Avoid making foam. If too much tissue stays at the bottom of the homogenizer carefully take out the pestle and use a disposable Pasteur pipette to bring the sample up into the medium. Expect a fairly homogeneous mixture, with no foam.
Alternatively, the sample can be ground with liquid nitrogen.After the last 1X PBS wash, place the tissue on a clean Petri dish and pat dry with a lint-free tissue. Transfer the sample to a precooled mortar and freeze with liquid nitrogen. Do 3 cycles of grind/freeze until you obtain a fine powder (like icing sugar). Transfer the powdered sample to a 100 mL glass beaker containing a magnetic stirrer and 30 mL NEB-complete buffer. Stir gently until an homogeneous mixture is obtained. Proceed with step 7.If you have access to other grinding equipment, like a bead beater or a TissueLyzer with a grinding jar, follow the instrument's instructions.
?. [Nuclei isolation]
Place one sheet of Miracloth inside the funnel and place it in a 50 mL Falcon tube. Pre-wet the Miracloth with 2-3 mL NEB complete. Discard the filtered solution.
?. [Nuclei isolation]
Filter the homogenized sample through the Miracloth sheet. If the flow is too slow, gently brush the inside of the sheet with a clean spatula. Discard the Miracloth sheet and adjust the volume to .
[ice cold NEB complete]
?. [Nuclei isolation]
Add  by pipetting it at the top of tube’s inside wall, in a circular way. Close the tube and mix genlty by invertion, about 20 times. The final concentration of Triton X-100 is 0.5%.
[25% Triton X-100]
?. [Nuclei isolation]
Collect the nuclei by centrifugation.
[Nuclei pellet]
[Nuclei pellet]
?. [Nuclei isolation]
Discard supernatant and gently resuspend the pellet with  by gently inverting the closed tube.
[ice cold NEB –βME]
Dispose the supernatant containing β-mercaptoethanol according to your institution's guidelines.
?. [Nuclei isolation]
Spin down as before. Discard the supernatant and resuspend the nuclei pellet in  by gently swirling the tube and keep on ice.
[Nuclei pellet]
[NEB_A]
?. [Percoll® gradients]
While waiting for the two centrifugations described above, prepare the Percoll® gradients:Prepare  and split into two 15 mL Falcon tubes. The two tubes should weigh exactly the same.Prepare 10 mL of  37.5% , split into two 15 mL Falcon  tubes. The two tubes should weigh exactly the same.
[75% Percoll® in NEB_A]
[37.5% Percoll® in NEB_A]
The buffers used for preparing the Percoll® gradients do not contain PVP-K40. In plants rich in polyphenols PVP can form a brown precipitate in the final agarose plug that prevents the DNA to migrate freely out of the agarose matrix.
?. [Percoll® gradients]
Assemble two 50 mL Falcon tubes with the 100 and 40 µm sieves, respectively, and place securely in on the ice bucket.
?. [Percoll® gradients]
Pour the nuclei suspension  through the 100 µm sieve, and then through the 40 µm sieve. Discard the sieves. Keep sample on ice.
[Nuclei suspension in NEB-A]
If the flow through the 40 μm sieve slows down, replace it with a new sieve.
?. [Percoll® gradients]
Collect nuclei as before . Discard supernatant and resuspend the nuclei pellet in  7 mL NEB_A. Repeat this step three more times.
[Nuclei pellet]
[NEB_A]
?. [Percoll® gradients]
Carefully overlay  over each 75% Percoll solution. The loaded tubes should weigh exactly the same.
[filtrated nuclei suspension]
?. [Percoll® gradients]
Collect the nuclei at the Percoll® gradient interphase by centrifugation . Adjust the acceleration = 9 (max), and deceleration = 1 (min).
[Percoll gradient]
[Percoll gradient]
?. [Percoll® gradients]
With a light source behind the gradient, collect the sample found at the interphase of the Percoll® gradient using a glass Pasteur pipette. Do this step very slowly. Transfer sample to a new 15 mL Falcon tube and keep on ice.
?. [Percoll® gradients]
Carefully overlay half of the sample on each 37.5% Percoll® tube. Each tube should weight exactly the same. Collect the nuclei by centrifugation  .
[Percoll gradient]
?. [Percoll® gradients]
Remove the solution with a disposable Pasteur pipette without disturbing the nuclei pellet, which is small and has a pale yellowish color.
?. [Percoll® gradients]
Resuspend each nuclei pellet in 10 mL NEB_B and collect by centrifugation. . Both acceleration and deceleration set at 9 (max).
[Nuclei wash after gradients]
[Nuclei wash after gradients]
?. [Percoll® gradients]
Remove the solution with a disposable Pasteur pipette without disturbing the nuclei pellet but leave ~  of solution. Gently tap the bottom of the tube to resuspend the nuclei and transfer to one 1.5 mL microcentrifuge tube. Repeat the same with the other nuclei pellet. Add ~  and spin down at 6,000 rpm, 1 min at room temperature.
200 µl
[NEB_B]
?. [Percoll® gradients]
Discard  and resuspend the nuclei in the remaining  by tapping the bottom of the tube. Keep on ice until ready.
[supernatant]
[NEB_B]
?. [Agarose plugs]
While running the Percoll® gradients, place the CleanCut™ 2% Agarose bottle in the water bath at . When the agarose is completely melted, transfer  to a screw-capped tube, and leave it in the water bath until ready.Precool one strip of disposable plug molds by sitting on a piece of Parafilm placed over the ice bucket.
70 °C
400 µl
?. [Agarose plugs]
Incubate the nuclei at , and the melted 2% agarose for .
43 °C
[nuclei + agarose mix]
?. [Agarose plugs]
Transfer the nuclei mix to the agarose tube, mix very gently by pipetting in and out until completely even.
?. [Agarose plugs]
Place precooled plug mold on the bench, over a paper towel.
?. [Agarose plugs]
Dispense ~  per plug. Prepare ~ 8 plugs. Cool down completely  .
90 µl
[Plugs setting]
[Plugs setting]
?. [Agarose plugs]
Pop out the agarose plug with the plastic tab at the end of the plug mold into the Proteinase K lysis mix at  without shaking.
[Proteinase K digestion]
[Proteinase K digestion]
?. [Agarose plugs]
Wash plugs in  at  for  without shaking.
[Wash-1]
50 °C
?. [Agarose plugs]
Wash plugs in  at  for  in a rotisserie at slowest speed. Place tube horizontally. The tube will rotate on its side, like a cabin of a Ferris wheel.
[Wash-2]
20 °C
?. [Agarose plugs]
Store each plug in ~ in screw capped tubes and store at  . Avoid introducing air bubbles in the tube.
[Wash-2]
4 °C
?. [Agarose plugs]
Analyze plugs by PFGE: 1% agarose gel (Certified™ Megabase Agarose BioRad) in 1X TAE. Settings: 60-120”, 120°, 6 V/cm, , . Stain gel with SYBR™ Safe.
15 °C