Lidstrom:QIAquick Gel Extraction Kit Protocol

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Cautions about gel purification

• Gel purificiation results in much lower yield than the manufacturers suggest.
  • They claim ~80+% recovery, but if you measure the amount before and after you will find you are lucky to recover 10%. Also, what is eluted is not pure. Something washes off of the columns that absorbs at ~A230nm.

  • 

    100uL of PCR was split into two; 25uL was purified with the Qiagen PCR purification protocol and the rest was run through a 0.7% agaose gel then gel purified. The red line represents the gel purified DNA and clearly has contaminant(s) from the gel and column process.

• There are alternative metods available, though not commonly applied:
  • freeze & squeeze kits
  • running the DNA band out of the gel using your gel running box (blog post)
    • JM loves the idea of cutting a hole in a gel and getting the DNA band you want to purify to run into the buffer in that well. So cool! If we had the modern gel running boxes with blue light illumination, I would be all over this.

QIAquick Gel Extraction Kit Protocol

From Marina via Aaron

• using a microcentrifuge
• This protocol is designed to extract and purify DNA of 70 bp to 10 kb from standard or low-melt agarose gels in TAE or TBE buffer. Up to 400 mg agarose can be processed per spin column. This kit can also be used for DNA cleanup from enzymatic reactions (see page 8). For DNA cleanup from enzymatic reactions using this protocol, add 3 volumes of Buffer QG and 1 volume of isopropanol to the reaction, mix, and proceed with step 6 of the protocol. Alternatively, use the MinElute Reaction Cleanup Kit.

Important points before starting

• The yellow color of Buffer QG indicates a pH 7.5.
• Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
• All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional table-top microcentrifuge at room temperature.

Procedure

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
   1. Minimize the size of the gel slice by removing extra agarose.
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 μl).
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation.
   1. IMPORTANT: Solubilize agarose completely.
4. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).
   1. If the color of the mixture is orange or violet, add 10 μl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow. The adsorption of DNA to the QIAquick membrane is efficient only at pH 7.5. Buffer QG contains a pH indicator which is yellow at pH ≤7.5 and orange or violet at higher pH, allowing easy determination of the optimal pH for DNA binding.
5. Add 1 gel volume of isopropanol to the sample and mix.
   1. For example, if the agarose gel slice is 100 mg, add 100 μl isopropanol. l ExtractionSpin Protocol
6. Place a QIAquick spin column in a provided 2 ml collection tube.
7. To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min.
   1. The maximum volume of the column reservoir is 800 μl. For sample volumes of more than 800 μl, simply load and spin again.
8. Discard flow-through and place QIAquick column back in the same collection tube.
   1. Collection tubes are reused to reduce plastic waste.
9. Recommended: Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1 min.
   1. This step will remove all traces of agarose. It is only required when the DNA will subsequently be used for direct sequencing, in vitro transcription, or microinjection.
10. To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min.
    1. Note: If the DNA will be used for salt-sensitive applications, such as blunt-end ligation and direct sequencing, let the column stand 2–5 min after addition of Buffer PE, before centrifuging.
11. Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).
    1. IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.
12. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
13. To elute DNA, add 25 μl ddH2O (pH=7.0) let the column stand for 1 min, and then centrifuge for 1 min.