Team:Newcastle/Minipreps

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(New page: ==Minipreps== ===Gel extraction=== #Excise the DNA fragment from the agarose gel with a clean, sharp scalpel #Weigh the gel slice in the colourless tube. Add 3 volumes Buffer QG to 1 vol...)
(Procedures)
 
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==Minipreps==
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{{Team:Newcastle/mainbanner}}
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===Gel extraction===
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=Minipreps using the Qiagen kit=
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#Excise the DNA fragment from the agarose gel with a clean, sharp scalpel
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==Materials required==
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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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* Eppendorf tubes
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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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* Pipettes
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#After the gel slice has dissolved completely, check the colour of the mixture is yellow
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* Appropriate overnight cultures
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#Add 1gell volume of isopropanol to the sample and mix
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* Buffer P1 (In the fridge)
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#Place QG quick spin column in 2ml collection tube
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* Buffer P2
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#Add sample and spin for 1 minute (discard flow through)
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* Buffer N3
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#Add QG buffer and spin for 1min (discard flow through)
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* Buffer PB
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#Add PE buffer and spin for 1min (discard flow through)(13000rpm)
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* Buffer EB
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#Place spin column in clean tube(1.5ml)
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* QIAprep spin column
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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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==Procedures==
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# Overnight culture should have been done the day before. Refer to [[Team:Newcastle/Growing an overnight cultures| growing an overnight culture]].
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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 minutes 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 30-60 seconds. Discard the flow-through.
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# Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60 seconds. Discard the flow-through.
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# Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30-60 seconds. 
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# Discard the flow-through, and centrifuge for an additional 1 minute 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 minute, and centrifuge for 1 minute.
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'''Go back to our [[Team:Newcastle/Protocol list|Protocol List]]'''
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{{Team:Newcastle/footer}}

Latest revision as of 12:54, 2 August 2010

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Minipreps using the Qiagen kit

Materials required

  • Eppendorf tubes
  • Pipettes
  • Appropriate overnight cultures
  • Buffer P1 (In the fridge)
  • Buffer P2
  • Buffer N3
  • Buffer PB
  • Buffer EB
  • QIAprep spin column

Procedures

  1. Overnight culture should have been done the day before. Refer to growing an overnight culture.
  2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
  3. Add 250 μl Buffer P2 and mix throughly by inverting the tube 4-6 times.
  4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
  5. Centrifuge for 10 minutes at 13,000 rpm in a table-top microcentrifuge.
  6. Apply the supernatant to the QIAprep spin column by decanting or pipetting.
  7. Centrifuge for 30-60 seconds. Discard the flow-through.
  8. Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60 seconds. Discard the flow-through.
  9. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30-60 seconds.
  10. Discard the flow-through, and centrifuge for an additional 1 minute to remove residual wash buffer.
  11. 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 minute, and centrifuge for 1 minute.


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