This protocol is designed to isolate and purify DNA of a certain length (say from a restriction digest or a PCR with secondary banding). Revised from the QIAquick Quick-Start Protocol.
- QIAquick Gel Extraction Kit
- Spin column tubes
- Razor blade
- 1.5mL epis
- Clean the transilluminator and a sharp razor blade by wiping repeatedly with ethanol. Don a lab coat (taping the collar and the wrists shut) , UV glasses, and a face shield and close the PCR room door before starting.
- Excise the DNA fragment of the desired size by cutting it out carefully with the razor blade and then flipping it on its sides and trimming off the extra gel. Then slice your band into a few smaller pieces and put in an epi. Wipe the razor blade off with ethanol and a kimwipe occasionally. If you are isolating multiple bands, slice off the lane you want to work with and set the rest of the gel aside out of the UV. You don't want to expose DNA to UV for more than a minute or so.
- Weigh your gel fragments (zeroing out the scale using an empty epi). They should be about 0.100g each.
- Add 3 volumes of Buffer QG to 1 volume of gel fragment (100 mg of gel ~ 100uL) and incubate in the 55C oven for about 10 minutes, vortexing every 2-3 minutes until the gel fragment is completely dissolved. If the mixture is orange or violet, add 10uL of 3M sodium acetate, pH 5.0 and mix. The mixture should turn yellow.
- Add 1 gel volume (about 100uL) of isopropyl and mix
- Place a QIAquick spin column in a provided 2 ml collection tube.
- To bind DNA, apply the sample to the QIAquick column and centrifuge for 30 seconds at 8000rpm (note: using lower spin times and speeds for the first few steps can minimize loss of DNA).
- Pour flow through back onto filter and spin again.
- Discard flow through into designated buffer waste container. Place the QIAquick column back in the same tube. Collection tubes are re-used to reduce plastic waste.
- To wash, add 0.75 ml Buffer PE to the QIAquick column. Let sit for 2-5 minutes to remove as much salt as possible before centrifuging for 30 seconds at 8000rpm.
- Discard flow through into buffer waste container and place the QIAquick column back in the same tube. Centrifuge column for an additional 1 minute at 13,000rpm. IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow through is discarded before this additional centrifugation.
- Remove filter column and check the sides for any remaining ethanol. Re-spin if necessary. Place filter column in a clean 1.5 ml microcentrifuge tube.
- To elute DNA, add 30 microliters hot Milli-Q water (heated to 65 degrees Celsius) to the center of the QIAquick membrane and let sit 2-5 minutes. Centrifuge the column for 2 minutes at 13,000rpm. Store DNA at -20 degrees Celsius as DNA may degrade in absence of buffering agent. (EB buffer is provided in the kit, but we use Milli-Q instead as the extra salt can inhibit ligation and sequencing reactions.)
Original protocol by: C. Eshelman Date: 13 Sept 12
Protocol Updated by: ___ Date: ___
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