Team:Mexico-UNAM-CINVESTAV/Protocols

From 2010.igem.org

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[[Image:miniprep.jpg]]
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Alkaline Lysis Mini Plasmid Preps
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#Grow O/N in 1.5 ml LMM or Terrific broth (see Reagents) with 75µg/ml Amp
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#Pour into ependorf tube and spin down cells at 7-8K for 2 min
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3. Aspirate s/n and resuspend in 50µl 25mM Tris pH 8, 10 mM EDTA; leave lids open
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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
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5. Add 75µl KoAc solution and vortex
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6. Add 100µl of phenol/CHI3, close lids, vortex
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7. Spin 13K for 2 mins
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8. Remove supernatant, add to 500µl ethanol. Vortex and spin at 13 K for 5 min
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9. Aspirate s/n, removing all ethanol
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10. Resuspend in 50µl TE
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11. Digest 2-5ul, adding 1µl preboiled 10mg/ml RNAse A (see Reagents for preparation).
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KOAc solution:
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- 60 ml 5M potassium acetate
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- 11.5 ml glacial acetic acid
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- 28.5 ml DDW

Revision as of 18:38, 26 October 2010

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

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