Difference between revisions of "Team:SKLMT-China/Notebook Protocol"

• use your fresh E.coli culture

• Put the cells on ice (and keep them on ice whenever possible).

• Spin the cells down at 9,000 rpm for 30 sec in the cooling centrifuge at 2°C.

• Discard the supernatant by decanting. Discard as much supernatant as possible.

• Resuspend the pellet in 1 ml of ice cold water by pipetting.

• Spin the cells at 10,000 rpm for 30 sec in the cooling centrifuge at 2°C.

• Discard the supernatant by decanting. Discard as much supernatant as possible.

• Resuspend the pellet in 1 ml of ice cold water by pipetting.

• Spin the cells down at 11,000 rpm for 30 sec in the cooling centrifuge at 2°C.

• Discard the supernatant by decanting and leave about 30 µl.

• Use the cells immediately.

• Add DNA to the cells and pipette the mixture into the chilled 1 mm electroporation cuvette.

• Set the electroporator to 1350 V, 10 µF, 600 Ohms. (This setting belongs to an Eppendorf® Electroporator 2510 using an electroporation cuvette with a gap of 1 mm. Other devices can be used, but the voltage has to be fixed at 1350V and the length of the pulse should be 5 ms.)

• Carefully knock the cuvette on the table to remove air bubbles and dry the metallic sides of the cuvette with a tissue. Do not touch the metallic sides with your hands.

• Place the cuvette into the holder of the electroporator, insert, and push the “pulse” button twice.

• Add 1 ml LB medium without antibiotics to the cuvette. Resuspend the cells carefully by pipetting up and down and pipette back into the reaction tube (avoid air bubbles in the suspension).

• Recover the cultures on an shaking incubator.

• (For future experiments in your lab, prepare a backup plate or backup tubes before starting, buffers are from Qiagen)

• Spin down the cells at 13,200 rpm for 1 minute.

• Discard the supernatant (remove as much as possible).

• Add 200 µl of P1 buffer (keep P1 at 4°C because of the RNaseA in the buffer).

• Resuspend the pellet by vortexing or use a pipette.

• Add 200 µl of P2 buffer (do not place P2 on ice, doing so will cause precipitation).

• Mix the tubes by inversion 5 times.

• Do not allow the lysis to proceed for more than 5 minutes.

• Add 200 µl of P3 buffer (keep P3 at 4°C).

• Mix by inversion 5 times.

• Spin the tubes at 13,200 rpm for 20 min.

• Prepare a fresh labeled 1.4 ml tube for each sample and add 500 µl of isopropanol .

• After centrifugation take out the supernatant with a pipette- avoiding white precipitate- and put it into the tube containing isopropanol.

• Mix by shaking the tubes vigorously.

• Spin the tubes at 13,200 rpm for 20 min.

• Discard the supernatant, invert the tube on a tissue.

• Carefully add 500 µl of 70% ethanol (centrifuge again for 5 min if pellet is only loosely attached to the wall of the tube).

• Discard the ethanol, invert the tube on a tissue.

• You may spin the tube again and remove remaining ethanol with a pipette.

• Dry the pellet for 10-15 min on a heating block (42C), do not overdry it (it is hard to dissolve then).

• For Plasmids: Dissolve the pellet in water (10-40 µl). For BACs: Dissolve the pellet in the digestion mix or dissolve in 8 µl water to send for sequencing.

• Transform the expression plasmid pSC101-BAD-gbaA-tet into the E.coli strain, in which you want to perform recombineering.

• To prepare DNA of the expression plasmid, using any commercial plasmid preparation kit will result in very low yield due to the low copy number of the pSC101 plasmid. Please use our plasmid preparation protocol. To check your prepared expression plasmid rather perform a function test than a digestion analysis.

• To start overnight cultures, pick colonies from the respective plate.

• Inoculate 1.5 ml reaction tubes containing 1.0 ml LB medium plus the appropriate antibiotics (select for BAC or plasmid and for expression plasmid)

• Puncture the cap for aeration.

• Incubate the cultures with shaking at 30°C and 950 rpm o/n.

• Set up punctured reaction tubes containing 1.4 ml fresh LB medium supplemented with the same antibiotics as in the ON culture.

• Inoculate each with 40µl fresh overnight culture.

• Incubate the tubes at 30°C, 950 rpm for 2 hours.

• Add 20 µl 10% L-Arabinose to a final concentration of 0.1%-0.2%. This will induce the expression of the proteins necessary for recombineering

• Leave some tubes without induction as negative controls

• Incubate at 37°C, 950 rpm for 40 min.

• Use 10% L-arabinose in dH2O, fresh or frozen in small aliquots at -20C

• Use 20 l stock solution per 1.4 ml LB for induction of recombination protein expression from pSC101-BAD-A-tet.