Latest revision as of 05:14, 16 February 2013
To purify your DNA from the agarose, you will use a kit from the Qiagen company. As we learned during the DNA extraction step on Day 2, reagents in such commercial kits can have uninformative names and their contents are in part proprietary.
- Estimate the volume of your gel slices by weighing them.
- The easiest way to do this is to pre-weigh an eppendorf tube for each slice, then weigh it again after adding the gel, and take the difference.
- What can you assume about the density of agarose and why?
- Add 3 volumes of QG for every 1 volume of agarose.
- The maximum advised volume is 550 μL. If you have a greater volume, continue for now, but first read step 6 to understand how to proceed later. Feel free to ask the teaching faculty for clarification.
- Incubate in the 50°C water bath for 10 minutes, until the agarose is completely dissolved. Every few minutes, you should remove your tubes from the 50°C heat and flick or vortex them for a few seconds to help dissolve the agarose.
- Add 1 volume — original gel volume, not current solution volume — of isopropanol to each eppendorf tube and pipet well to mix.
- Get two QIAquick columns and two collection tubes from the teaching faculty. Label the spin-columns (not the collection tubes!) with your sample IDs and then pipet the appropriate dissolved agarose mixture to the top of each one. Microfuge the column in the collection tube for 60 seconds at maximum speed (approx. 16,000 rcf). The maximum capacity of the QIAquick columns is 800 uL! If you have more than 800 uL in your mixture, you will need to repeat this step using the same column.
- Discard the flow-through in the sink and replace the spin-columns in their collection tubes. Add 500 μL of QG to the top of the column and spin as before.
- Discard the flow-through again, then add 750 μL of PE to the top of the column and incubate for 5 min at room temperature.
- Spin for 1 min as before.
- Discard the flow-through in a temporary waste container such as a 15 mL conical tube (PE contains ethanol) and replace the spin-columns in their collection tubes.
- Add nothing to the top but spin for 60 seconds more to dry the membrane.
- This step completely removes remaining ethanol that could interfere with future reactions.
- Trim the caps off of two new eppendorf tubes and prepare sticky labels (in your team color) for the top: write the date, your section day, and the sample ID. You may also want to label the side of each tube, so you don't lose track of which sample is which in the following step.
- Place the labeled spin-column in its matching trimmed eppendorf tube and add 30 μL of pH 7 water to the center of the membrane.
- Do not add regular distilled water, as this is at a lower pH, which will lower elution efficiency.
- Allow the columns to sit at room temperature for one minute and then spin as before. The material that collects in the bottom of the eppendorf tubes is your purified 16S DNA.