plasmid isolation, alkaline lysis (minprep)

introduction

The isolation of plasmids from bacteria, is one of  the simplest biochemical excersize in a molecular biological lab. It is a beautiful illustartion where the pH and differences in solubility of molecules are used to separate them from each other. The wole procedure can be done in about 30 min, quick and clean. The method is desribed in Birnboim and Doly, (1979) and also on wikipedia.

method

  • grow an overnite culture (about 2 mL)
  • next morning, pour a 1.5 mL tube, keep the rest for a possible glycerol later
  • spin 1 min at max speed
  • pour of the sup
  • resuspend pellet in 100 ul Solution I (grate the tube close over a tube rack for speed)
  • add 200 ul Solution II (there is a 5 min incubation step here, to lyse the cells, but if you are impatient, the bacteria should be lysed in a min when mixed well, just look for non turbidity)
  • add 150 ul Solution III, mix well, incubate 5 min (again, shorter should be no problem, but also think about that coffee, it is not going to drink itself now is it)
  • spin 5 min, leave the pellet (preciptated proteins and genomic DNA) and pipette ~450 ul clear sup to a new tube
  • add 1 mL 95% EtOH, mix spin 5 min
  • remove sup, wash pellet with 70% EtOH, remove wash, dry pellet
  • resuspend in 25-50 ul 0.1 ug/ul RNAse A sol (I add it here, so the RNA can function as a carrier for the plasmid DNA)
  • whoop, done!

solutions

Solution I (for 50 mL)
50 mM Glucose (0.45 g)
25 mM Tris-HCl (1.25 ml 1 M Tris-Cl, pH 8)
10 mM EDTA (1 ml 0.5 M EDTA, pH 8)

Solution II (for 50 ml)
1% SDS (0.5 g SDS)
0.2 M NaOH (1 ml 10 M NaOH)

Solution III (for 50 ml)
3 M KAc (14.7 g; Mw=98.15)
pH to 5.5 with 100% HAc

RNAse A sol (0.1 ug/ul in water)

 

Discussion

When I was an undergrad, I used a ‘kit’ for this experiment, because our lab was not really a molecular biological lab, but later when I needed to set-up molecular biological courses for undergrads, the practicum coordinator laughed out loud when I asked for the ‘kit’. That’s not only a waste of money, but also of time and intellect! I love the smell of a miniprep in the morning!

Advantages of good-old optimized method over the kit

  1. cheaper
  2. faster (sure, drying the pellet takes time, but don’t you have some coffee to drink?)
  3. I hate those little plastic columns, it is impossible to get them out of the centrifuge 2 at the time
  4. yield is higher, not limited by binding  capacity of the membrane
  5. up-scaling is easy, just grown more and multiply the volumes, and divide over more tubes
  6. the DNA is clean, I mean, how clean do you need it to be, I can sequence from these plasmids, that is as good as it needs to be. I am not microinjecting cells or something, I am sure then you need to do aditional steps
  7. here’s my claim that while in London, I most definately achieved the world record in plasmid isolation, w/o robots, or multiwell plates. I did 48 preps in under one hour, time stared when I poured the first bacterial culture in the first tube, and stopped when I pipetted water on the last pellet [official record-breaking attempts will be notified here, let me know]

reference

Birnboim HC, Doly J (November 1979). “A rapid alkaline extraction procedure for screening recombinant plasmid DNA“. Nucleic Acids Res. 7 (6): 1513–23
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