Notebook

Protocols

DNA MIDI prep

Solutions
TE buffer pH 8.0
STE buffer: 25% Sucrose, 50mM Tris/HCl pH8, 10mM EDTA
STET buffer: 8% Sucrose, 50mM Tris pH8, 50mM EDTA, 5% Trition 100X
Lysozyme powder or solution: 20µL sol., stock 100mg/mL, 50% glycerol (store at -20°C)
PhenolChoroform pH8

Cultivate overnight, then retake on
Liquid: on 50mL of LB+Antibiotic
Solid: spread 100µL of solution on a petri Dish with conveniant ATB

Retake culture
Liquid: Centri. Vmax 5’, retake pellet on 2 mL STE buffer
Solid: Retake colony on a COREX tube after add of 1mL STE

Plasmidic DNA obtention
· Add 2mg of lysozyme, vortex then incubate 2’ (room temp.)
· Add 5mL of STET, invert tightly and incubate 10’ (room temp.)
· WarmBath at 100°C, 1’30’’ (4 tubes per time) until white color appears (the transfer in centri. tube)
· Centri. 11000-17000 RPM 15’, keep supernatant
· Filtrate pellet for retaken maximum amount of supernatant (without white)
· +5mL Isopropanol, mix by inverting
· Centri 11000-17000 RPM 5’, discard supernatant
· Dry out the tube by using absorbing paper
· Retake pellet in 500µL TE ph8, transfer into eppendorfs
· +20µL RNAse (10mg/mL)
· Incubate 15’, 45°C
· Incubate 15’, 65°C
· +50µL NaCl 5M
· +500µL PhenolCholoform ph8
· Vortex then discard the 2nd phase (phase of the top = Water with DNA)
· Centri. Vmax 2’, discard the second phase, repeat operation until you retake the most of top-phase
· Precipitate DNA with 1mL of EtOH 100%
· Centri. Vmax, 1’
· Wash 2 times with 200µL
· Let pellet dry out
· Resuspend in 50µL of TE