Team:Newcastle/Qiagen Minipreps

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Contents

Minipreps using the Qiagen kit

Plasmid extraction

Materials required

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  • 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.

QIAquick Gel Extraction Microcentrifuge Protocol

Materials

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  1. Scalpel
  2. Eppendorf tubes
  3. Pipettes
  4. QIAquick column
  5. UV transluminator
  6. Buffer QG
  7. Buffer PE
  8. Buffer EB
  9. Isopropanol
  10. 70% ethanol

Protocol

  1. Before extraction, clean the UV transilluminator and scalpel with 70% ethanol.
  2. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. ( Minimise the exposure of the gel to UV as much as possible.)
  3. Weight the gel slice and add 3 volumes of buffer QG to 1 volume of gel (100 mg ~ 100 µl).
  4. Incubate at 50°C and invert the tube gently at regular interval until the gel has completely dissolved.
  5. After the gel has dissolved completely, check that the color of the mixture is yellow.
  6. Add 1 gel volume of isopropanol to the sample and mix.
  7. Place a QIAquick spin column in a 2 ml collection tube
  8. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min.
  9. Discard the flow through and place the QIAquick column back into the same tube (max volume: 750 µl).
  10. Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1 min. Discard the flow through and place the QIAquick column back into the same tube.
  11. To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min. Place the QIAquick column back into the same tube.
  12. Centrifuge the column for a further 1 min.
  13. Transfer the column into a clean 1.5 ml microcentrifuge tube.
  14. To elute DNA, add 30 µl of Buffer EB to the center of the QIAquick membrane and allow to stand for 1 min.
  15. Centrifuge the column for 1 min and transfer the eluate to a clean 1.5 ml micriocentrifuge tube.
  16. To measure the purity of the sample, use a Nanodrop Spectrophotometer.
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