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

From 2010.igem.org

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(New page: ==Qiagen Mini-prep plasmid isolation== This protocol is based on QIAGEN® Plasmid Purification Handbook. This protocol is designed for preparation of up to 20 µg of high-copy plasmid or ...)
 
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==Qiagen Mini-prep plasmid isolation==
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=Qiagen Mini-prep plasmid isolation=
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 20 µg of high-copy plasmid or cosmid DNA using the Qiagen Plasmid Mini Kit.
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This protocol is designed for preparation of up to 20 µg of high-copy plasmid or cosmid DNA using the Qiagen Plasmid Mini 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 20:
Maximum recommended culture volumes for the Qiagen-tip 20:
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High-copy plasmids   5 mL
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High-copy plasmids   1-5 mL
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1. Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2–5 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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2. Harvest the 5 mL bacterial cells by centrifugation at 4.000 rpm for 15 min at 4°C (blue cap 15 mL tube). If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C.
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''Materials:''
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3. Resuspend the bacterial pellet in 0.3 mL of chilled Buffer P1 (containing RNAse).
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- bacterial culture
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4. Add 0.3 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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- Qiagen colums
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5. Add 0.3 mL of chilled Buffer P3, mix gently by inverting 4–6 times, and incubate on ice for 5 minutes.
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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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6. Centrifuge 10 min at 4.000 rpm and pour supernatant through cheesecloth into a fresh blue cap 50 mL tube.
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- buffer P2 (200 mM NaOH, 1% SDS)
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7. During centrifugation equilibrate a Qiagen-tip 20 by applying 1 mL Buffer QBT, and allow the column to empty by gravity flow.
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- buffer P3 (3 M KAc, pH 5.5)
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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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- buffer PE
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9. Wash the Qiagen-tip with 2 × 2 mL Buffer QC.
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- milliQ pH 8.0
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10. Elute DNA with 0.8 mL Buffer QF into a clean 15 mL blue cap tube.
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- centrifuge
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11. Precipitate DNA by adding 0.56 mL (0.7 volumes) room-temperature isopropanol to the eluted DNA.
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- nanodrop
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12. Mix and centrifuge immediately at 4.000 rpm for 30 minutes at 4 °C.
 
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13. Proceed immediately when centrifuge stops and carefully decant the supernatant.
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''Protocol:''
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14. Wash DNA pellet with 1 mL of cold 70% ethanol, and centrifuge at 4.000 rpm for 10 minutes at 4 °C.
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1. Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2–5 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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15. Carefully decant the supernatant without disturbing the pellet.
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2. Harvest the 5 mL bacterial cells by centrifugation at 13,000 rpm for 1 min at 20°C (microcentrifuge tube). If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C
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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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3. Resuspend pelleted bacterial cells in 250 μL Buffer P1. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.  
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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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4. Add 250 μL Buffer P2 and mix thoroughly by inverting the tube 4–6 times. Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 minutes.
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5. Add 350 μL Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture volumes (e.g. ≥5 mL) may require inverting up to 10 times. The solution should become cloudy.
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Used buffers:
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6. Incubate at -20 °C for 15 minutes.
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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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7. Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge. A compact white pellet will form.
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Buffer P2: 200 mM NaOH, 1% SDS (Make fresh, every two weeks)
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8. Apply the supernatants from step 7 to the QIAprep spin column by decanting or pipetting.
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Buffer P3: 3 M KAc, pH 5.5
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9. Centrifuge for 30–60 seconds. Discard the flow-through.
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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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10. Wash QIAprep spin column by adding 0.75 mL Buffer PE and centrifuging for 30–60 seconds.
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Buffer QC: 1 M NaCl, 50 mM MOPS, 15% Ethanol, pH 7.0
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11. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
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Important: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.
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Buffer QF: 1.25 M NaCl, 50 mM Tris/HCl, 15% Ethanol, pH 8.5
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12. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 30 μL Buffer EB (10 mM Tris•Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 minute, and centrifuge for 1 minute.
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TE buffer: 10 mM Tris/HCl, 0.1 mM EDTA, pH 8.0
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13. Measure DNA concentration on the Nanodrop.

Latest revision as of 11:03, 12 September 2010

Qiagen Mini-prep plasmid isolation

This protocol is based on QIAGEN® Plasmid Purification Handbook. This protocol is designed for preparation of up to 20 µg of high-copy plasmid or cosmid DNA using the Qiagen Plasmid Mini Kit. Qiagen plasmid isolation is used to obtain very pure plasmid DNA.

Maximum recommended culture volumes for the Qiagen-tip 20:

High-copy plasmids 1-5 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 PE

- milliQ pH 8.0

- centrifuge

- nanodrop


Protocol:

1. Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2–5 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 5 mL bacterial cells by centrifugation at 13,000 rpm for 1 min at 20°C (microcentrifuge tube). If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C

3. Resuspend pelleted bacterial cells in 250 μL Buffer P1. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.

4. Add 250 μL Buffer P2 and mix thoroughly by inverting the tube 4–6 times. Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 minutes.

5. Add 350 μL Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture volumes (e.g. ≥5 mL) may require inverting up to 10 times. The solution should become cloudy.

6. Incubate at -20 °C for 15 minutes.

7. Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge. A compact white pellet will form.

8. Apply the supernatants from step 7 to the QIAprep spin column by decanting or pipetting.

9. Centrifuge for 30–60 seconds. Discard the flow-through.

10. Wash QIAprep spin column by adding 0.75 mL Buffer PE and centrifuging for 30–60 seconds.

11. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer. Important: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.

12. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 30 μL Buffer EB (10 mM Tris•Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 minute, and centrifuge for 1 minute.

13. Measure DNA concentration on the Nanodrop.

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