TITLE: ILT3
AUTHORS: Mar Roca Cugat, UM ILT Team

[DESCRIPTION]
-

[STEPS]
SECTION: Bacterial cell destruction
1. Resuspend the bacterial pellet in 0.3 mL of Resuspension Buffer.
SECTION: Bacterial cell destruction
1.1. Mix vigorously using the vortex until no clumps remain. 
The solution needs to be “clear” not “turbid” otherwise invert the tube a few times more and continue.
The lysate should appear viscous. Do not allow the lysis reaction to proceed for more than 5 min.
There may be little treads seen by opening the tube when you start step 3. That is the genomic DNA!
From now on, the bacteria are lysed so no GMO any more (lysed bacteria are no bacteria any more). However, you need to continue working under MLI conditions as the whole laboratory is MLI!
SECTION: Bacterial cell destruction
2. Add 0.3 mL of Lysis Buffer, mix thoroughly by vigorously inverting the sealed tube for 10 times.
SECTION: Bacterial cell destruction
3. Add 0.3 mL of chilled Neutralisation Buffer, mix immediately and thoroughly by vigorously shaking 10 s.
If the mixture still appears viscous, more mixing is required to completely neutralize the solution.
SECTION: Plasmid recovery
4. Centrifuge at maximum speed in a centrifuge for 2 min.
Do not disturb the pellet. You can stick your pipette directly through the potentially small white top layer. The white stuff that may stick to the outside of the pipet is no problem as long as you do not transfer this into the new tube, only all the liquid is needed.
SECTION: Plasmid recovery
5. Collect 700 µL the supernatant in a 1.5 ml microcentrifuge tubes.
Do not disturb the pellet. You can stick your pipet directly through the potentially small white top layer. The white stuff that may stick to the outside of the pipet is no problem as long as you do not transfer this into the new tube, only all the liquid is needed.

SECTION: Plasmid recovery
6. Precipitate DNA by adding 700 µL isopropanol to the plasmid DNA containing supernatant. Mix vigorously using the vortex.

SECTION: Plasmid recovery
7. Centrifuge at maximum speed in a centrifuge for 7 min.
Always place the tubes with the hinge of the lit pointed outwards so you know where the pellet should be if you cannot see it in the next steps.

SECTION: Plasmid recovery
8. Pipet away all the fluid. take care NOT to touch or suck up the pellet (your plasmids).
Be care full not to touch your pellet or suck it up. (don't scratch the bottom with your pipet tip) Leave the lowest/last 10 microliters there. no need to remove them now.
SECTION: Plasmid recovery
9. Add 1 mL 70 % (v/v) ethanol to the tube with the plasmid pellet, close the tube and vortex shortly (10 s).

SECTION: Plasmid recovery
10. Centrifuge at maximal speed for 3 min.

SECTION: Plasmid recovery
11. Remove the 70% ethanol by pipetting.
Use a 200 microliter pipet tip instead of the blue 1000 microliter tips. Pipet the last remaining microliters qualitatively. Do this really carefully to not disturb the plasmid DNA pellet.
SECTION: Plasmid recovery
12. Air dry for 5 min
If the pellet just turns from white "opaque" it’s already ok! Please set a timer to not over dry the pellet 5 minutes should be ok to evaporate the ethanol the remaining little droplets are water.
SECTION: Plasmid recovery
13. Add 50 µL sterile water and vortex.
If the vortexing results in scattering all the water though out the tube shorty centrifugate to collect the water with plasmids again. The plasmids are now in solution and will stay in solution regardless the centrifugation.

SECTION: Restriction enzyme digestion (D2)
14. Make the mixtures for the restriction analysis, a total of 20 µL :
  5 µL 
  2 µL 
  1 µL 
  12 µL
SECTION: Restriction enzyme digestion (D2)
14.1. Mix components by pipetting the reaction mixture up and down, or by “flicking” the reaction tube. Follow with a quick (“touch”) spin-down in a microcentrifuge.
Do not vortex the reaction too vigorously -this may kill the restriction enzyme).
Do not discard the remaining plasmid product! You need to analyze this as well!
Do not forget to add the enzyme as last component.
SECTION: Restriction enzyme digestion (D2)
15. Label a reaction tubes and place them in the heating block. Digestion will take place at 37 °C for 30 min.

SECTION: Restriction enzyme digestion (D2)
16. Add DNA gel loading dye to each sample to stop the reaction.
SECTION: Agarose gel preparation
17. Prepare the casting tray by putting the sides together.
SECTION: Agarose gel preparation
18. The silicon coated site should point towards the inside.
SECTION: Agarose gel preparation
19. Apply the elastic band around the extruding parts in the middle of the sides to hold everything in place.
SECTION: Agarose gel preparation
20. Flip the assembled tray in the correct position and press it lightly on the table to ensure proper alignment of the both sides.
SECTION: Agarose gel preparation
21. Add the comb and put the tray in the box to catch any potential spills if not correctly assembled.
SECTION: Agarose gel preparation
22. Prepare a 1% agarose gel in 1x TAE buffer. The total volume depends on the size of the casting tray; we will make 100 mL gels. Calculate and weigh the amount of agarose required for your volume. Deposit the agarose in a glass Erlenmeyer flask and add the indicated volume of 1x TAE buffer.
SECTION: Agarose gel preparation
23. Pour the mixture in the casting tray with combs. Remove any air bubbles with a plastic filter tip.


SECTION: Agarose gel preparation
24. Allow the gel to solidify (10 min). 

SECTION: Agarose gel preparation
24.1. Remove the comb.
SECTION: Agarose gel preparation
25. Insert the casting tray into the electrophoresis equipment; the side of the slots in the direction of the cathode (black). 
Do not add the metal casting system into the electrophorese system, just the gel.
SECTION: Agarose gel preparation
26. Prepare and add 1x TAE Buffer to the system until the gel is completely submerged.
27.