Barney:PCR purification

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Materials

Qiagen QiaQuick Spin columns

Qiagen Buffer PB

OR
5M Guanidine Hydrochloride
10mM Tris-HCl pH 6.6
30% ethanol

Qiagen Buffer PE

OR
10mM Tris-HCl pH 7.5
70% ethanol

Qiagen Buffer EB

OR
10mM Tris-HCl pH 8.5


DNAse-free Milli-Q Water for elution (should be slightly basic, pH 7.0-8.5)

1M HCl

Protocol

kit manual

  1. Bind DNA
    1. Add 5 volumes of Buffer PB (or equivalent) to PCR reaction to be purified (i.e. add 250μL to a 50μL PCR reaction)
    2. Load onto a clean spin column, place the column in a clean collection tube, cap, and centrifuge (5000rpm, 1 minute)
    3. Discard flow-through, place column back in empty collection tube
  2. Wash
    1. Add 700μL of Buffer PE (or equivalent) to column and centrifuge (13000rpm, 1 minute)
    2. Discard flow-through
    3. Replace empty column in collection tube and centrifuge again (13000rpm, 1 minute) to dry column
  3. Elution for Sequencing
    1. add 30-50μL of DNAse-free Milli-Q water to the center of the column
    2. incubate for 1 minute at room temperature
    3. place column into new, sterile DNAse-free tube for DNA collection
    4. centrifuge (13000rpm, 1 minute)
  4. Elution for downstream processing
    1. add 30-50μL of Buffer EB to the center of the column
    2. incubate for 1 minute at room temperature
    3. place column into new, sterile DNAse-free tube for DNA collection
    4. centrifuge (13000rpm, 1 minute)


Column Re-Use Protocol

  1. step 1
  2. step 2
  3. step 3
  4. etc.
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