Team:Newcastle/Gel extraction

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(New page: ====QIAquick gel extraction microcentrifuge protocol==== # Excise the DNA fragment from the agarose gel with a clean, sharp scalpel # Weight the gel slice and add 3 volumes of buffer QG to...)
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====QIAquick gel extraction microcentrifuge protocol====
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# Excise the DNA fragment from the agarose gel with a clean, sharp scalpel
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# Weight the gel slice and add 3 volumes of buffer QG to 1 volume of gel (100 mg ~ 100 µl)
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=QIAquick Gel Extraction Microcentrifuge Protocol=
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# Incubate at 50°C and invert the tube gently at regular intrerval until the gel has completely dissolved
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# After the gel has dissovled completely, check that the color of the mixture is yellow
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==Materials==
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# Add 1 gel volume of isopropanol to the sample and mix
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# Scalpel
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# Place a QIAquick spin column in a 2 ml collection tube
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# Eppendorf tubes
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# To bind DNA, apple the sample to the QIAquick column and centrifuge for 1 min
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# Pipettes
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# Discard the flow through and place the QIAquick column back into the same tube (Maximum volumn is 750ul)
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# QIAquick column
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# Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1 min and discard the flow through and place the QIAquick column back into the same tube
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# UV transluminator
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# To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min and place the QIAquick column back into the same tube
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# Buffer QG
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# Centrifuge the column for a further 1 min  
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# Buffer PE
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# Transfer the column into a clean 1.5 ml micriocentrifuge tube
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# Buffer EB
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# To elute DNA, add 30 µl of Buffer EB to the center of the QIAquick membrane and allow to stand for 1 min  
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# Isopropanol
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# Centrifuge the column for 1 min and transfer the eluate to a clean 1.5 ml micriocentrifuge tube
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# 70% ethanol
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==Protocol==
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# Before extraction, clean the UV transilluminator  and scalpel with 70% ethanol.
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# 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.)
 +
# Weight the gel slice and add 3 volumes of buffer QG to 1 volume of gel (100 mg ~ 100 µl).
 +
# Incubate at 50°C and invert the tube gently at regular interval until the gel has completely dissolved.
 +
# After the gel has dissolved completely, check that the color of the mixture is yellow.
 +
# Add 1 gel volume of isopropanol to the sample and mix.
 +
# Place a QIAquick spin column in a 2 ml collection tube.
 +
# To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min.
 +
# Discard the flow through and place the QIAquick column back into the same tube (max volume: 750 µl).
 +
# 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.
 +
# 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.
 +
# Centrifuge the column for a further 1 min.
 +
# Transfer the column into a clean 1.5 ml microcentrifuge tube.
 +
# To elute DNA, add 30 µl of Buffer EB to the center of the QIAquick membrane and allow to stand for 1 min.
 +
# Centrifuge the column for 1 min and transfer the eluate to a clean 1.5 ml micriocentrifuge tube.
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# To measure the purity of the sample, use a [[TeamNewcastleNanoDrop Spectrophotometer| Nanodrop Spectrophotometer]].
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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 14:26, 26 October 2010

iGEM Homepage Newcastle University BacillaFilla Homepage Image Map

QIAquick Gel Extraction Microcentrifuge Protocol

Materials

  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.
Newcastle lab 4.jpeg
Newcastle lab 5.jpeg
Newcastle lab 6.jpeg
Newcastle lab 7.jpeg
Newcastle lab 8.jpeg

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