IGEM:Brown/2007/Lab Protocols/10min E coli Miniprep: Difference between revisions

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(New page: Stocks: TENS: for 100ml: 1ml 10N NaOH 1ml 20% SDS 1ml 1M Tris pH 7.5 200 microliters 0.5 M EDTA ...)
 
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Procudure:
Procudure:


1. Spin 1.5 ml o/n culture E coli in an eppendorf for 10 s
# Spin 1.5 ml o/n culture E coli in an eppendorf for 10 s
2. Decant s/n and resuspend in the remaining volume
# Decant s/n and resuspend in the remaining volume
3. Add 300 microliters TENS. Vortex to mix completely
# Add 300 microliters TENS. Vortex to mix completely
4. Add 150 microliters 3 M NaOAC. Mix by inversion
# Add 150 microliters 3 M NaOAC. Mix by inversion
5. Spin 10 min room temperature (debris, genomic DNA)
# Spin 10 min room temperature (debris, genomic DNA)
6. Transfer s/n to fresh tube
# Transfer s/n to fresh tube
7. Add 900 microliters ice cold EtOH. Mix thoroughly
# Add 900 microliters ice cold EtOH. Mix thoroughly
8. Sping 15 min room temperature
# Sping 15 min room temperature
( 9. Wash pellet 1x 70% EtOH )
( # Wash pellet 1x 70% EtOH )
10. Dry pellet speed-vac
# Dry pellet speed-vac
11. Resuspend in 100 microliter TE. Use 1.5-3 microliters for digest
# Resuspend in 100 microliter TE. Use 1.5-3 microliters for digest
12. Add 1 microliter1 1mg/ml RNAase to 10 microliter digest
# Add 1 microliter1 1mg/ml RNAase to 10 microliter digest

Revision as of 09:16, 7 June 2007

Stocks: TENS: for 100ml: 1ml 10N NaOH 

                          1ml 20% SDS
                          1ml 1M Tris pH 7.5
                          200 microliters 0.5 M EDTA
                          96.8 mL water
        3 M NaOAc pH 5.2

Procudure:

  1. Spin 1.5 ml o/n culture E coli in an eppendorf for 10 s
  2. Decant s/n and resuspend in the remaining volume
  3. Add 300 microliters TENS. Vortex to mix completely
  4. Add 150 microliters 3 M NaOAC. Mix by inversion
  5. Spin 10 min room temperature (debris, genomic DNA)
  6. Transfer s/n to fresh tube
  7. Add 900 microliters ice cold EtOH. Mix thoroughly
  8. Sping 15 min room temperature

( # Wash pellet 1x 70% EtOH )

  1. Dry pellet speed-vac
  2. Resuspend in 100 microliter TE. Use 1.5-3 microliters for digest
  3. Add 1 microliter1 1mg/ml RNAase to 10 microliter digest
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