McGill/Midiprep Seeding

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Revision as of 21:59, 3 June 2007

Protocol

1. Set up 50ml of 1xLB with 100 ug/ml antibiotic of your choice.
   1. Pen-strep should be at 100mg/ml stock so use 50ml per 50 ml.
   2. Kanamycin should be at 10mg/ml (it's poorly soluble) so use 500 ul per ml NOTE - for low copy number plasmids, increase total culture volume to 100ml.
2. Distribute the LB/antibiotic mix into 50ml-tubes (25ml in each). This gives room to aerate.
3. Add 250ul overnight culture (from a 5ml prep) to each tube, incubate on the shaker at 37C overnight.
4. Pellet the bacteria in a desktop clinical centrifuge, 10-15 min. at medium setting should do it. (You should have that kind of centrifuge on the C-floor, it might take either 50ml tubes or 15ml tubes seem to be more efficient. Hint - if you don't see a pellet it was too short/too slow. These guys will settle out at room temperature eventually, so if you have a wimpy centrifuge don't worry, just add more time.
5. Decant supernatant, drain well, pool all pellets of the same type into 5ml buffer P1. Make sure RNAse A and LyseBlue (included) were added to P1. Distribute the mix along 10 microfuge tubes, 500ul each.
6. To each microfuge tube, add 500ul buffer P2, mix by inverting several times (do not vortex). Do one tube at a time (add, mix, go to next tube, add mix...). You know it's mixed when it turns from white/beige to uniformly blue - no white streaks allowed.
7. Leave at room temperature 5 minutes. By the time you get to tube 10, it may be close for the time to start the next step (#8) for tube 1.
8. Add 50ul buffer P3, mix by inverting as above. Do one tube at a time, same order as above. A white precipitate should form and all the blue should disappear.
9. Leave tubes on ice for 10 min.
10. Centrifuge tubes in a microfuge at max speed for 10 min.
11. Pool all supernatants into a 15ml tube. Pipet with P1000, do not pour, do not be greedy (ie. take liquid only, no white chunks).
12. If there are chunks, re-aliquot into clean microfuge tubes. There will be fewer tubes than before. Re-centrifuge, pool supernatants into another clean 15ml tube. If there are no chunks, go on to step 13.
13. Pre-equilibrate a midiprep column with 5ml QBT buffer.
14. When all the buffer has gone through, pour on your cleared bacterial lysate from step 12.
15. When all the sample has gone through, pour the column full of QC Buffer (no need to measure, just fill right to the top). Allow to drain, repeat once.
16. When all the QC buffer has gone through, MOVE THE COLUMN TO A CLEAN 50ml TUBE, then apply 5ml buffer QF. THIS STEP ELUTES THE DNA.
17. Add 3.5ml isopropanol to the eluted DNA, distribute among six microfuge tubes, centrifuge at max speed for 10 min.
18. Decant supernatant, pipet off any remaining liquid with a p-10 tip, air dry 10 min.
19. Resuspend in EB or TE or in 10mM Tris pH8.0, recommended volumes: tiny pellet (barely visible) 25ul, medium pellet 50ul, huge pellet 100uL.

• To measure the O.D. 260/280, usually 4ul in 400ul water is ok.