Barney:PCR purification

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.