Alkaline Lysis Miniprep protocol

From 2007.igem.org

(Difference between revisions)
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#Inoculate 5mL of LB medium with appropriate antibiotic (e.g. 5 microliter of ampicillin) with a single bacterial colony and incubate at 37 degrees C overnight with vigorous shaking.
#Inoculate 5mL of LB medium with appropriate antibiotic (e.g. 5 microliter of ampicillin) with a single bacterial colony and incubate at 37 degrees C overnight with vigorous shaking.
-
 
#Pour 1.5mL of the above culture into an Eppendorf tube. Centrifuge for 1 min. at 10,000g and discard the supernatant by aspiration/decanting.
#Pour 1.5mL of the above culture into an Eppendorf tube. Centrifuge for 1 min. at 10,000g and discard the supernatant by aspiration/decanting.
-
 
-
 
#Resuspend the pellet by vortexing  in 100ul of ice-cold Solution A:  
#Resuspend the pellet by vortexing  in 100ul of ice-cold Solution A:  
50mM Glucose
50mM Glucose
10mM EDTA
10mM EDTA
25mM Tris Cl (pH 8.0)
25mM Tris Cl (pH 8.0)
-
 
#Add 200 microliters of freshly prepared Solution B:
#Add 200 microliters of freshly prepared Solution B:
0.2M NaOH
0.2M NaOH
Line 44: Line 40:
invert rapidly 4 times. No vortexing.
invert rapidly 4 times. No vortexing.
-
 
#Add 150 microliters of ice-cold Solution C:
#Add 150 microliters of ice-cold Solution C:
3M potassium
3M potassium
5M acetate
5M acetate
(pH 4.8)
(pH 4.8)
-
 
#vortex for 10 seconds and place on ice for 15 minutes.
#vortex for 10 seconds and place on ice for 15 minutes.
-
 
#Transfer the supernatant to a fresh tube.
#Transfer the supernatant to a fresh tube.
-
 
#Add an equal amount (400ul) of phenol/chroloform (200/200ul).
#Add an equal amount (400ul) of phenol/chroloform (200/200ul).
Vortex briefly and centrifuge at 10,000g for 2 minutes.
Vortex briefly and centrifuge at 10,000g for 2 minutes.
-
 
#Transfer the supernatant to a fresh tube.
#Transfer the supernatant to a fresh tube.
-
 
#Add two volumes (800ul) of 100% ethanol. Vortex and place on ice for 20 minutes.
#Add two volumes (800ul) of 100% ethanol. Vortex and place on ice for 20 minutes.
Centrifuge at 10,000g for 15 minutes. Discard the supernatant.
Centrifuge at 10,000g for 15 minutes. Discard the supernatant.
-
 
#Wash by adding 400ul of 70% ethanol. Centrifuge for 10 minutes.
#Wash by adding 400ul of 70% ethanol. Centrifuge for 10 minutes.
Discard the supernatant and allow the tube to dry (e.g. vacuum dessicator).
Discard the supernatant and allow the tube to dry (e.g. vacuum dessicator).
-
 
#Add 30ul of TE buffer (or water) with a small amount of RNAase.
#Add 30ul of TE buffer (or water) with a small amount of RNAase.

Revision as of 12:54, 28 September 2007

<Return to list of protocols> <Team home page>

  • Applications:
    1. Purification of plasmid DNA from bacterial cultures
  • Time to complete protocol:
    • Lab time: 3h
    • Waiting time: 30min, 15min, 1hour, overnight.
  • Approximate cost of materials: $

Contents

Method from primary and secondary reagents

Primary & secondary Reagents Required including controls
  • Bacterial colony
  • appropriate antibiotic
  • LB (cupboard)
  • EDTA
  • Glycerol
  • TRIS
  • NaOH
  • SDS
  • Potassium Acetate
  • Phenol
  • Chloroform
  • 100% Ethanol
  • 70% Ethanol
  • milliQ water
  • RNAse
Method including controls
  1. Inoculate 5mL of LB medium with appropriate antibiotic (e.g. 5 microliter of ampicillin) with a single bacterial colony and incubate at 37 degrees C overnight with vigorous shaking.
  2. Pour 1.5mL of the above culture into an Eppendorf tube. Centrifuge for 1 min. at 10,000g and discard the supernatant by aspiration/decanting.
  3. Resuspend the pellet by vortexing in 100ul of ice-cold Solution A:

50mM Glucose 10mM EDTA 25mM Tris Cl (pH 8.0)

  1. Add 200 microliters of freshly prepared Solution B:

0.2M NaOH 1% SDS

invert rapidly 4 times. No vortexing.

  1. Add 150 microliters of ice-cold Solution C:

3M potassium 5M acetate (pH 4.8)

  1. vortex for 10 seconds and place on ice for 15 minutes.
  2. Transfer the supernatant to a fresh tube.
  3. Add an equal amount (400ul) of phenol/chroloform (200/200ul).

Vortex briefly and centrifuge at 10,000g for 2 minutes.

  1. Transfer the supernatant to a fresh tube.
  2. Add two volumes (800ul) of 100% ethanol. Vortex and place on ice for 20 minutes.

Centrifuge at 10,000g for 15 minutes. Discard the supernatant.

  1. Wash by adding 400ul of 70% ethanol. Centrifuge for 10 minutes.

Discard the supernatant and allow the tube to dry (e.g. vacuum dessicator).

  1. Add 30ul of TE buffer (or water) with a small amount of RNAase.
Equipement Required
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