Team:TU Delft/protocols/birnboim plasmid isolation

From 2010.igem.org

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(New page: ''Protocol'' 1. Pour 1 mL overnight bacterial culture into Eppendorf tube, centrifuge for 1 minute at maximum speed (13,000 rpm). Carefully suck off supernatant 2. Resuspend the bacteria...)
 
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''Protocol''
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=Birnboim plasmid isolation=
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1. Pour 1 mL overnight bacterial culture into Eppendorf tube, centrifuge for 1 minute at maximum speed (13,000 rpm). Carefully suck off supernatant
 
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2. Resuspend the bacterial pellet in 100 μL cold solution I, suspend well (vortex) and incubate for 15 min on ice
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This plasmid isolation is used to obtain plasmid DNA that can be used to test the transformed colonies for the right insert.
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3. Vortex shortly and add immediately 200 μL fresh solution II (up to two weeks old, not cold), mix vigorously (no vortexing) and leave 1 minute at room temperature
 
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4. Add 150 μL cold solution III, mix gently until white precipitate appears and incubate on ice for 30 minutes
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''Materials:''
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5. Add 40 μL chloroform, and mix gently (optional)
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- bacterial culture
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6. Centrifuge at maximum speed for 5 minutes at room temperature (Eppendorf centrifuge)
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- milliQ
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7. Take 400 μL of the supernatant (beware of taking along some pellet), put in a clear eppendorf tube and add to 800 μL of cold (-20 °C) 100% ethanol
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- BB solution I (10 mg/mL glucose; 25 mM Tris-HCl pH 8,0; 10 mM EDTA, 0,1 mg/mL RNAse A)
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8. Mix, leave at -20 °C for 30 minutes and centrifuge at room temperature for 10 minutes maximum speed (Eppendorf centrifuge)
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- BB solution II (0.2 NaOH; 1% SDS
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9. Carefully pipet off the supernatant
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- BB solution III (3 M NaAc, pH 4,8  = 100 mL: 40,8 g NaAc.3H2O, 38 mL HAc, 52 mL H2O)
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10. Add 100 μL solution IV, mix by vortexing and add 200 μL ice cold 100% ethanol. Incubate at -20 °C for 30 minutes. Centrifuge 10 minutes full speed (Eppendorf centrifuge), carefully discard supernatant (pipet)
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- BB solution IV (0.3 m NaAc)
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11. Wash DNA pellet with 1 mL of ice cold 70% ethanol and centrifuge for 10 minutes maximum speed (Eppendorf centrifuge) can carefully pipet off the supernant
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- cold 70% ethanol
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- cold 100% ethanol
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- microcentrifuge
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- nanodrop
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''Protocol:''
 +
 
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1. Pour 1 mL overnight bacterial culture into Eppendorf tube, centrifuge for 1 minute at maximum speed (13.000 rpm). Carefully suck off supernatant
 +
 
 +
2. Resuspend the bacterial pellet in 100 μL cold solution I, suspend well (vortex) and incubate for  15 min on ice
 +
 
 +
3. Vortex shortly and add immediately 200 μL fresh solution II (make fresh, every two weeks, keep at room temperature), mix vigorously (no vortexing) and leave 1 minute at room temperature
 +
 
 +
4. Add 150 μL cold solution III, mix gently until white precipitate appears and incubate on ice for 30 minutes
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12. Air-dry the pellet for 5 minutes, and redissolve the DNA in 50 μL water
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5. Centrifuge at maximum speed for 5 minutes at room temperature (Eppendorf centrifuge)
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13. Measure DNA concentration on the Nanodrop
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6. Take 400 μL of the supernatant (beware of taking along some pellet) and add to 800 μL of cold (-20 °C) 100% ethanol
 +
7. Mix, leave at -20 °C for 30 minutes and centrifuge at room temperature for 10 minutes maximum speed (Eppendorf centrifuge)
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''Used buffers:''
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8. Carefully pipet off the supernatant
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BB solution I: 10 mg/mL glucose; 25 mM Tris-HCl pH 8,0; 10 mM EDTA,
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9. Add 100 μL solution IV, mix by vortexing and add 200 μL ice cold 100% ethanol. Incubate at -20 °C for 30 minutes. Centrifuge 10 minutes full speed (Eppendorf centrifuge), carefully discard supernatant (pipet)
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0,1 mg/mL RNAse A, freshly added (keep at 4 °C)
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BB solution II: 0,2 NaOH; 1% SDS (keep at RT; make fresh, every two weeks)
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10. Wash DNA pellet with 1 mL of ice cold 70% ethanol and centrifuge for 10 minutes maximum speed (Eppendorf centrifuge) can carefully pipet off the supernatant
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BB solution III: 3 M NaAc (pH 4,8) 
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11. Air-dry the pellet for 5 minutes, and redissolve the DNA in 50 μL water
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(= 100 mL: 40,8 g NaAc 3 H2O, 38 mL HAc, 52 mL H2O)
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(check pH; keep at 4 °C)
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BB solution IV: 0,3 m NaAc
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12. Measure DNA concentration on the Nanodrop

Latest revision as of 09:30, 12 September 2010

Birnboim plasmid isolation

This plasmid isolation is used to obtain plasmid DNA that can be used to test the transformed colonies for the right insert.


Materials:

- bacterial culture

- milliQ

- BB solution I (10 mg/mL glucose; 25 mM Tris-HCl pH 8,0; 10 mM EDTA, 0,1 mg/mL RNAse A)

- BB solution II (0.2 NaOH; 1% SDS

- BB solution III (3 M NaAc, pH 4,8 = 100 mL: 40,8 g NaAc.3H2O, 38 mL HAc, 52 mL H2O)

- BB solution IV (0.3 m NaAc)

- cold 70% ethanol

- cold 100% ethanol

- microcentrifuge

- nanodrop


Protocol:

1. Pour 1 mL overnight bacterial culture into Eppendorf tube, centrifuge for 1 minute at maximum speed (13.000 rpm). Carefully suck off supernatant

2. Resuspend the bacterial pellet in 100 μL cold solution I, suspend well (vortex) and incubate for 15 min on ice

3. Vortex shortly and add immediately 200 μL fresh solution II (make fresh, every two weeks, keep at room temperature), mix vigorously (no vortexing) and leave 1 minute at room temperature

4. Add 150 μL cold solution III, mix gently until white precipitate appears and incubate on ice for 30 minutes

5. Centrifuge at maximum speed for 5 minutes at room temperature (Eppendorf centrifuge)

6. Take 400 μL of the supernatant (beware of taking along some pellet) and add to 800 μL of cold (-20 °C) 100% ethanol

7. Mix, leave at -20 °C for 30 minutes and centrifuge at room temperature for 10 minutes maximum speed (Eppendorf centrifuge)

8. Carefully pipet off the supernatant

9. Add 100 μL solution IV, mix by vortexing and add 200 μL ice cold 100% ethanol. Incubate at -20 °C for 30 minutes. Centrifuge 10 minutes full speed (Eppendorf centrifuge), carefully discard supernatant (pipet)

10. Wash DNA pellet with 1 mL of ice cold 70% ethanol and centrifuge for 10 minutes maximum speed (Eppendorf centrifuge) can carefully pipet off the supernatant

11. Air-dry the pellet for 5 minutes, and redissolve the DNA in 50 μL water

12. Measure DNA concentration on the Nanodrop

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