Team:TU Delft/protocols/midi-prep plasmid isolation

From 2010.igem.org

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(New page: ==Qiagen Midi-prep plasmid isolation== This protocol is based on QIAGEN® Plasmid Purification Handbook. This protocol is designed for preparation of up to 100 µg of high- or low-copy p...)
 
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==Qiagen Midi-prep plasmid isolation==
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=Qiagen Midi-prep plasmid isolation=
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This protocol is based on QIAGEN® Plasmid Purification Handbook.
This protocol is based on QIAGEN® Plasmid Purification Handbook.
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This protocol is designed for preparation of up to 100 µg of high- or low-copy plasmid or cosmid DNA using the Qiagen Plasmid Midi Kit.
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This protocol is designed for preparation of up to 100 µg of high- or low-copy plasmid or cosmid DNA using the Qiagen Plasmid Midi Kit. Qiagen plasmid isolation is used to obtain very pure plasmid DNA.
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Qiagen plasmid isolation is used to obtain very pure plasmid DNA.
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Maximum recommended culture volumes for the Qiagen-tip 100:
Maximum recommended culture volumes for the Qiagen-tip 100:
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1. Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 25 to 100 mL LB medium containing the appropriate selective antibiotic. Incubate for approximately 8 h at 37°C with vigorous shaking (approx. 300 rpm).
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''Materials:''
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- bacterial culture
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- Qiagen colums
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- buffer P1 (100 mg/mL RNAse A, 50 mM Tris/HCl, 10 mM EDTA, pH 8.0)
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- buffer P2 (200 mM NaOH, 1% SDS)
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- buffer P3 (3 M KAc, pH 5.5)
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- buffer QBT (750 mM NaCl, 50 mM MOPS, 15% Ethanol, pH 7.0, 0.15% Triton X-100)
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2. Harvest the 25 to 100 mL bacterial cells by centrifugation at 4.000 rpm for 15 min at 4°C ( Sorvall buckets). If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C.
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- buffer QC (1 M NaCl, 50 mM MOPS, 15% Ethanol, pH 7.0)
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3. Resuspend the bacterial pellet in 4 mL of chilled Buffer P1 (containing RNAse) and pour into a blue cap 50 mL tube.
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- buffer QF (1.25 M NaCl, 50 mM Tris/HCl, 15% Ethanol, pH 8.5)
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4. Add 4 mL of Buffer P2, mix gently but thoroughly by inverting 4–6 times, and incubate at room temperature for 5 minutes.
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- isopropanol
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5. Add 4 mL of chilled Buffer P3, mix gently by inverting 4–6 times, and incubate on ice for 15 or 20 minutes.
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- milliQ
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6. Centrifuge 30 min at 4.000 rpm and pour supernatant through cheesecloth into a fresh blue cap 50 mL tube.
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- centrifuge
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7. During centrifugation equilibrate a Qiagen-tip 100 by applying 4 mL Buffer QBT, and allow the column to empty by gravity flow.
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- nanodrop
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8. Apply the supernatant from step 6 onto the Qiagen-tip and allow it to enter the resin by gravity flow.
 
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9. Wash the Qiagen-tip with 2 × 10 mL Buffer QC.
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''Protocol:''
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10. Elute DNA with 5 mL Buffer QF into 15 mL blue cap tube.
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1. Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 25 to 100 mL LB medium containing the appropriate selective antibiotic. Incubate for approximately 8 h at 37°C with vigorous shaking (approx. 300 rpm)
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11. Precipitate DNA by adding 3.5 mL (0.7 volumes) room-temperature isopropanol to the eluted DNA.
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2. Harvest the 25 to 100 mL bacterial cells by centrifugation at 4.000 rpm for 15 min at 4°C ( Sorvall buckets). If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C
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12. Mix and centrifuge immediately at 4.000 rpm for 30 minutes at 4 °C.
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3. Resuspend the bacterial pellet in 4 mL of chilled Buffer P1 (containing RNAse) and pour into a blue cap 50 mL tube
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13. Proceed immediately when centrifuge stops and carefully decant the supernatant.
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4. Add 4 mL of Buffer P2, mix gently but thoroughly by inverting 4–6 times, and incubate at room temperature for 5 minutes
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14. Wash DNA pellet with 2 mL of cold 70% ethanol, and centrifuge at 4.000 rpm for 10 minutes at 4 °C.
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5. Add 4 mL of chilled Buffer P3, mix gently by inverting 4–6 times, and incubate on ice for 15 minutes
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15. Carefully decant the supernatant without disturbing the pellet.
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6. Centrifuge 30 min at 4.000 rpm and pour supernatant through cheesecloth into a fresh blue cap 50 mL tube
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16. Air-dry the pellet for 5–10 min (has to be dry), and redissolve the DNA in a suitable volume of buffer (e.g.,
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7. During centrifugation equilibrate a Qiagen-tip 100 by applying 4 mL Buffer QBT, and allow the column to empty by gravity flow
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TE buffer pH 8.0). Normally 100 mL TE buffer or autoclaved milliQ H2O was used.
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17. Measure DNA concentration on the Nanodrop
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8. Apply the supernatant from step 6 onto the Qiagen-tip and allow it to enter the resin by gravity flow
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9. Wash the Qiagen-tip with 2 × 10 mL Buffer QC
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Used buffers:
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10. Elute DNA with 5 mL Buffer QF into 15 mL blue cap tube
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Buffer P1: 100 mg/mL RNAse A, 50 mM Tris/HCl, 10 mM EDTA, pH 8.0  
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11. Precipitate DNA by adding 3.5 mL (0.7 volumes) room-temperature isopropanol to the eluted DNA
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Buffer P2: 200 mM NaOH, 1% SDS (Make fresh, every two weeks)
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12. Mix and centrifuge immediately at 4.000 rpm for 30 minutes at 4 °C
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Buffer P3: 3 M KAc, pH 5.5
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13. Proceed immediately when centrifuge stops and carefully decant the supernatant
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Buffer QBT: 750 mM NaCl, 50 mM MOPS, 15% Ethanol, pH 7.0, 0.15% Triton X-100
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14. Wash DNA pellet with 2 mL of cold 70% ethanol, and centrifuge at 4.000 rpm for 10 minutes at 4 °C
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Buffer QC: 1 M NaCl, 50 mM MOPS, 15% Ethanol, pH 7.0
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15. Carefully decant the supernatant without disturbing the pellet
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Buffer QF: 1.25 M NaCl, 50 mM Tris/HCl, 15% Ethanol, pH 8.5
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16. Air-dry the pellet for 5–10 min (has to be dry), and redissolve the DNA in a suitable volume of milliQ H2O
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TE buffer: 10 mM Tris/HCl, 0.1 mM EDTA, pH 8.0
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17. Measure DNA concentration on the Nanodrop.

Latest revision as of 11:05, 12 September 2010

Qiagen Midi-prep plasmid isolation

This protocol is based on QIAGEN® Plasmid Purification Handbook. This protocol is designed for preparation of up to 100 µg of high- or low-copy plasmid or cosmid DNA using the Qiagen Plasmid Midi Kit. Qiagen plasmid isolation is used to obtain very pure plasmid DNA.

Maximum recommended culture volumes for the Qiagen-tip 100:

High-copy plasmids 25 mL

Low-copy plasmids 100 mL


Materials:

- bacterial culture

- Qiagen colums

- buffer P1 (100 mg/mL RNAse A, 50 mM Tris/HCl, 10 mM EDTA, pH 8.0)

- buffer P2 (200 mM NaOH, 1% SDS)

- buffer P3 (3 M KAc, pH 5.5)

- buffer QBT (750 mM NaCl, 50 mM MOPS, 15% Ethanol, pH 7.0, 0.15% Triton X-100)

- buffer QC (1 M NaCl, 50 mM MOPS, 15% Ethanol, pH 7.0)

- buffer QF (1.25 M NaCl, 50 mM Tris/HCl, 15% Ethanol, pH 8.5)

- isopropanol

- milliQ

- centrifuge

- nanodrop


Protocol:

1. Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 25 to 100 mL LB medium containing the appropriate selective antibiotic. Incubate for approximately 8 h at 37°C with vigorous shaking (approx. 300 rpm)

2. Harvest the 25 to 100 mL bacterial cells by centrifugation at 4.000 rpm for 15 min at 4°C ( Sorvall buckets). If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C

3. Resuspend the bacterial pellet in 4 mL of chilled Buffer P1 (containing RNAse) and pour into a blue cap 50 mL tube

4. Add 4 mL of Buffer P2, mix gently but thoroughly by inverting 4–6 times, and incubate at room temperature for 5 minutes

5. Add 4 mL of chilled Buffer P3, mix gently by inverting 4–6 times, and incubate on ice for 15 minutes

6. Centrifuge 30 min at 4.000 rpm and pour supernatant through cheesecloth into a fresh blue cap 50 mL tube

7. During centrifugation equilibrate a Qiagen-tip 100 by applying 4 mL Buffer QBT, and allow the column to empty by gravity flow

8. Apply the supernatant from step 6 onto the Qiagen-tip and allow it to enter the resin by gravity flow

9. Wash the Qiagen-tip with 2 × 10 mL Buffer QC

10. Elute DNA with 5 mL Buffer QF into 15 mL blue cap tube

11. Precipitate DNA by adding 3.5 mL (0.7 volumes) room-temperature isopropanol to the eluted DNA

12. Mix and centrifuge immediately at 4.000 rpm for 30 minutes at 4 °C

13. Proceed immediately when centrifuge stops and carefully decant the supernatant

14. Wash DNA pellet with 2 mL of cold 70% ethanol, and centrifuge at 4.000 rpm for 10 minutes at 4 °C

15. Carefully decant the supernatant without disturbing the pellet

16. Air-dry the pellet for 5–10 min (has to be dry), and redissolve the DNA in a suitable volume of milliQ H2O

17. Measure DNA concentration on the Nanodrop.

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