Team:Newcastle/Minipreps

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==Minipreps==
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=Minipreps=
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===Gel extraction===
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==Procedures==
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The Miniprep Kit is used to extract plasmid.
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# Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
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# Add 250 μl Buffer P2 and mix throughly by inverting the tube 4-6 times.
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# Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
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# Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge.
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# Apply the supernatant to the QIAprep spin column by decanting or pipetting.
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# Centrifuge for 30-60s. Discard the flow-through.
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# Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60s. Discard the flow-through.
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# Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30-60s. 
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# Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
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# To elute DNA, place the QIAprep column in a clean 1.5 ml microcentrifuge tube. Add 50 μl Buffer EB or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.
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#Excise the DNA fragment from the agarose gel with a clean, sharp scalpel
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#Weigh the gel slice in the colourless tube. Add 3 volumes Buffer QG to 1 volume of gel slice in a colourless tube (100mg~10µl)
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#Incubate at 50°C for 10 min (or until the gel slice has completely dissolved)vortex every 2-3minutes
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#After the gel slice has dissolved completely, check the colour of the mixture is yellow
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#Add 1gell volume of isopropanol to the sample and mix
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#Place QG quick spin column in 2ml collection tube
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#Add sample and spin for 1 minute (discard flow through)
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#Add QG buffer and spin for 1min (discard flow through)
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#Add PE buffer and spin for 1min (discard flow through)(13000rpm)
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#Place spin column in clean tube(1.5ml)
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#Add elution buffer (50µl)spin for 1 min
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#If using a gel to analyse add loading dye and mix (pipet up and down)
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Revision as of 15:07, 28 July 2010

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Minipreps

Procedures

The Miniprep Kit is used to extract plasmid.

  1. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
  2. Add 250 μl Buffer P2 and mix throughly by inverting the tube 4-6 times.
  3. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
  4. Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge.
  5. Apply the supernatant to the QIAprep spin column by decanting or pipetting.
  6. Centrifuge for 30-60s. Discard the flow-through.
  7. Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60s. Discard the flow-through.
  8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30-60s.
  9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
  10. To elute DNA, place the QIAprep column in a clean 1.5 ml microcentrifuge tube. Add 50 μl Buffer EB or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.
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