IGEM:IMPERIAL/Protocols/maxi prep of culture

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Maxi prep – Purification of DNA

1) Check if the centrifuge in room 616 is free. 2) Transfer the O/N culture into centrifuge bottle. 3) Set up the rotor, making sure that two pins are perpendicular to each other. 4) After balancing, spin at 5,000 rpm, 4oC, code = 03 or 30. Spin for 5min. 5) Remove supernatant into the conical flask (dirty one and add Virkon and leave for 10 min). 6) Add 10ml of solution I into pellet and resuspend fully by vortexing. 7) Add 20ml of solution II (check for precipitation before adding.) and swirl. 8) Add 15ml of solution III and mix well by inversion. 9) Spin for 5min at 9,000 rpm at 4oC. 10) Place a piece of filter cloth on the centrifuge bottle and filter the supernatant through it. 11) Scrape the pellet into yellow bag, Wash thoroughly 12) Add 45ml propan-2-ol (isopropanol). NB: Isopropanol precipitates nucleic acid.

13) Mix well and spin again at the same condition as step 9. 14) Drain supernatant down the sink. Handle with care as the precipitant may be loose. 15) Leave to dry on a piece of tissue, up side down. 16) Add 3ml of MiliQ and dissolve the precipitant. 17) Add 4ml -20oC 5M LiCl and swirl. NB: LiCl precipitate out high MW RNA. 18) Aliquot into 5 eppendorfs. 19) Leave on ice for 5min (do not stop at this step, all other steps can be left whilst you do other things but not here). 20) Spin in the bench centrifuge for 5min at 13k rpm. 21) Collect supernatants into 50ml falcon tube which should come to approximately 7ml. 22) Add 14ml of 100% Ethanol. NB: Absolute ethanol precipitates nucleic acid. 23) Leave to stand on ice for 5min. 24) Spin with the centrifuge 5804Rm programme 2. 25) Remove supernatant and leave to dry, up side down. 26) Add 500ul MiliQ and resuspend. Transfer this into a fresh eppendorf. 27) Add 10ul of RNase A and leave for 15min on 37oC hot block. NB: RNase chops up small MW RNA into nucleotides. 28) Add 600ul 2.5M NaCl+20% PEG solution. Make sure this is mixed well before adding. 29) Stand on ice for 5min. 30) Spin for 5min at 13k rpm. 31) Remove supernatant, and spin again to remove all the residual liquid. 32) Resuspend in 500ul MiliQ by pipetting and vortexing. This may take a long time. 33) Add 500ul of phenol chloroform and vortex (hold tube and lid firmly). 34) Safety glasses should be worn. NB: Phenol is extremely dangerous it causes burns and it can be absorbed through skin and cause a cumulative damage to genetic material. If in contact, use 50% glycerol wash and then wash off with water. 35) Spin for 5min at 13k rpm, take top layer to new tube. 36) Add 500ul chloroform. 37) Vortex and spin for 3min at 13k rpm. 38) Take the aqueous fraction (i.e. top layer) into a fresh eppendorf. 39) Add 50ul 3M NaOAc, pH5.2 and 1ml of absolute ethanol. 40) Mix by inversions and spin for 5min, until DNA precipitant becomes visible. 41) Remove supernatant and spin again to remove residual liquid. 42) Resuspend in 100-300ul MiliQ.