Alkaline Lysis Miniprep protocol

From 2007.igem.org

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====Method from primary and secondary reagents====
====Method from primary and secondary reagents====
=====Primary & secondary Reagents Required including controls=====
=====Primary & secondary Reagents Required including controls=====
-
*Bacterial colony
+
*Bacterial colony overnight culture
*appropriate antibiotic
*appropriate antibiotic
*[[Melbourne/Secondary Reagent LB|LB]] (cupboard)
*[[Melbourne/Secondary Reagent LB|LB]] (cupboard)
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*[[Melbourne/primary glycerol|Glycerol]]
*[[Melbourne/primary glycerol|Glycerol]]
*[[Melbourne/primary Tris|TRIS]]
*[[Melbourne/primary Tris|TRIS]]
-
*NaOH
+
*[[Melbourne/primary NaOH|NaOH]]
*SDS
*SDS
*Potassium Acetate
*Potassium Acetate
*Phenol
*Phenol
*Chloroform
*Chloroform
-
*100% Ethanol
+
*100% [[Melbourne/primary Ethanol|Ethanol]]
-
*70% Ethanol
+
*70% [[Melbourne/primary Ethanol|Ethanol]]
*[[Melbourne/primary milliq|milliQ water]]
*[[Melbourne/primary milliq|milliQ water]]
*RNAse
*RNAse
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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: <BR>-50mM Glucose <BR>-10mM EDTA <BR>-25mM Tris Cl (pH 8.0)  
-
 
+
#Add 200 microliters of freshly prepared Solution B:<BR>-0.2M NaOH<BR>-1% SDS <BR> invert rapidly 4 times. No vortexing.
-
#Resuspend the pellet by vortexing  in 100ul of ice-cold Solution A:  
+
#Add 150 microliters of ice-cold Solution C: <BR> -3M potassium <BR> -5M acetate (pH 4.8)
-
50mM Glucose
+
-
10mM EDTA
+
-
25mM Tris Cl (pH 8.0)
+
-
 
+
-
#Add 200 microliters of freshly prepared Solution B:
+
-
0.2M NaOH
+
-
1% SDS
+
-
 
+
-
invert rapidly 4 times. No vortexing.
+
-
 
+
-
#Add 150 microliters of ice-cold Solution C:
+
-
3M potassium
+
-
5M acetate
+
-
(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). Vortex briefly and centrifuge at 10,000g for 2 minutes.
-
#Add an equal amount (400ul) of phenol/chroloform (200/200ul).
+
-
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. Centrifuge at 10,000g for 15 minutes. Discard the supernatant.
-
#Add two volumes (800ul) of 100% ethanol. Vortex and place on ice for 20 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).
-
Centrifuge at 10,000g for 15 minutes. Discard the supernatant.
+
-
 
+
-
#Wash by adding 400ul of 70% ethanol. Centrifuge for 10 minutes.
+
-
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.

Latest revision as of 11:11, 29 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
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)
  4. Add 200 microliters of freshly prepared Solution B:
    -0.2M NaOH
    -1% SDS
    invert rapidly 4 times. No vortexing.
  5. Add 150 microliters of ice-cold Solution C:
    -3M potassium
    -5M acetate (pH 4.8)
  6. vortex for 10 seconds and place on ice for 15 minutes.
  7. Transfer the supernatant to a fresh tube.
  8. Add an equal amount (400ul) of phenol/chroloform (200/200ul). Vortex briefly and centrifuge at 10,000g for 2 minutes.
  9. Transfer the supernatant to a fresh tube.
  10. 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.
  11. Wash by adding 400ul of 70% ethanol. Centrifuge for 10 minutes. Discard the supernatant and allow the tube to dry (e.g. vacuum dessicator).
  12. Add 30ul of TE buffer (or water) with a small amount of RNAase.
Equipement Required
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