DNA extraction from gel
- Prepare gel with a 1% concentration (i.e. 1.00 gr Agarose low melt point, 100 ml TAE 1X)
- Make the wells thicker than usual to fit more DNA into them (you want to maximise the DNA yield, hence minimise the agarose quantity)
- Run 1 hour at 90V
QIA quick spin Gel Extraction Kit procedure
- Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose.
- Weigh the eppendorf tubes BEFORE moving the agarose band to them so you can weigh it and assess the gel weight.
- Add 3 volumes of Buffer QG to 1 volume of gel (300μL ==> 100 mg)
- Incubate at 50° 10 minutes (vortexing every 2-3 minutes helps dissolve agarose)
- After the gel slice has dissolved completely, check that the color of the mixture is yellow, it it is NOT add 10 μL Sodium acetate 3M
- If your DNA fragment is <500 bp and >4 kb increase the yield by adding 1 gel volume of isopropanol to the sample 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 1 min.
The maximum volume of the column reservoir is 800 µl. For sample volumes of more than 800 µl, simply load and spin again.
- Discard flow-through and place QIAquick column back in the same collection tube
- (Optional): Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1 min. This is to remove all traces of agarose
- To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min. 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.
- Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at ≥10,000 x g (~13,000 rpm).
- Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
- To elute DNA, add 50 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of the QIAquick membrane and centrifuge the column for 1 min at maximum speed. For increased DNA concentration, add 30 µl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min.