Team:Mexico-UNAM-CINVESTAV/Protocols

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Miniprep.jpg


Alkaline Lysis Mini Plasmid Preps

  1. Grow O/N in 1.5 ml LMM or Terrific broth (see Reagents) with 75µg/ml Amp
  2. Pour into ependorf tube and spin down cells at 7-8K for 2 min
  3. Aspirate s/n and resuspend in 50µl 25mM Tris pH 8, 10 mM EDTA; leave lids open
  4. Add 100µl of freshly prepared 1% SDS, 0.2M NaOH (5ml = 100ul 10M NaOH added to 4.4ml DDW then 500µl 10% SDS). Add it forcefully and you don't need to vortex
  5. Add 75µl KoAc solution and vortex
  6. Add 100µl of phenol/CHI3, close lids, vortex
  7. Spin 13K for 2 mins
  8. Remove supernatant, add to 500µl ethanol. Vortex and spin at 13 K for 5 min
  9. Aspirate s/n, removing all ethanol
  10. Resuspend in 50µl TE
  11. Digest 2-5ul, adding 1µl preboiled 10mg/ml RNAse A (see Reagents for preparation).

KOAc solution: - 60 ml 5M potassium acetate

- 11.5 ml glacial acetic acid

- 28.5 ml DDW


DNA Isolation for Low-Melting Point Agarose (using elu-tip method)

  1. Excise fragment from gel and estimate volume.
  2. Add 1/100 volume of 1 M Tris pH 7.5, 1/50 volume of 0.5 M EDTA, and 1/100 volume of 5M NaCl.
  3. Incubate at 68°C for 10 minutes.
  4. Vortex and remove to a small tube.
  5. Incubate at 37°C for 5 minutes.
  6. Phenol extract 2-3 times (phenol at 42°C, spin at 13 K for 2 minutes.)
  7. Ether extract 1 time. Place tubes at 65°C, then speed-vac 2 to 5 minutes. Repeat procedure about 4 times to get rid of residual ether.
  8. Ethanol precipitate DNA and suspend pellet in 1 ml of low salt buffer (from elu-tip column protocol)
  9. Following the elu-tip protocol booklet, wash the column by pushing 5 ml of low salt buffer through the matrix at a rate of 0.5-1.0 ml/minute. The column may be incubated in the low salt buffer ³ 2 hours to improve recovery.
  10. Load DNA sample onto the column slowly (1-2 drops/second). NOTE: When recovering DNA from low-melt temperature agarose, use of the pre-filter is not recommended. Consult the protocols booklet for specific parameters of different types of nucleic acid purification (i.e. DNA purification when LMP agarose isn't used).
  11. Wash the column with 2-3 mls of pre-warmed (42°C) low salt buffer.
  12. Elude DNA with 0.4 ml of high salt buffer. Do a total of 2-3 washes as desired to be certain of good recovery.
  13. Ethanol precipitate DNA and resuspend in TE or dH2O.
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