Modified Gel Extraction Kit Protocol

This protocol is a modified version of the Qiagen Gel Extraction Kit protocol, used to purify PCR samples.
 * 1) Transfer samples to new micro-centrifuge tubes.
 * 2) Add 3-4 volumes of QG buffer to each sample and mix by vortexing.
 * 3) Transfer to provided spin column and let stand for 2 minutes.
 * 4) Centrifuge for 30 seconds at 13,000 rpm.
 * 5) Pour the liquid back into the filter column and let stand for 2 minutes.
 * 6) Centrifuge for 30 seconds at 13,000 rpm.
 * 7) Discard the liquid.
 * 8) Add 750 uL of PE buffer to the filter and let stand for 2 minutes.
 * 9) Centrifuge for 30 seconds at 13,000 rpm.
 * 10) Discard the liquid.
 * 11) Add another 750 uL of PE buffer and let stand for 2 minutes.
 * 12) Centrifuge for 30 seconds at 13,000 rpm.
 * 13) Discard the liquid.
 * 14) Centrifuge for 1 minute at 13,000 rpm.
 * 15) Incubate with filter cap open for 1 minute at 65-70 degrees Celsius.
 * 16) Transfer filter to a new micro-centrifuge tube.
 * 17) Add 30-50 uL of elution buffer to the filter.
 * 18) Incubate with filter cap closed for 4 minutes at 65-70 degrees Celsius.
 * 19) Centrifuge for 1 minute at 0.5 x g.
 * 20) Centrifuge for 2 minutes at 13,000 rpm.
 * 21) Measure concentrations of DNA in eluant with NanoDrop.
 * 22) Repeat the elution steps if necessary.

Some helpful suggestions:

Put the PE buffer in the -20 degree freezer when starting the procedure so that it is cold when you need to use it. This prevents the DNA from being washed out of the filter during steps 8-13. Cold ethanol in the PE buffer will not take the DNA with it, but room temperature may reduce final concentrations.

Remember to give your PE buffer a couple inversions before use to make sure it is mixed thoroughly.

Incubating the filter column with the cap open will evaporate some of the ethanol on the filter. Be careful not to incubate for too long with the cap open, as it may dry out the filter and cause DNA to adhere too much.

Centrifuging at a low speed during step 19 will help any evaporated elution buffer to settle back into the filter before the final spin at 13,000 rpm.

You may use samples that contain loading dye. It has not been found yet that it effects the binding of DNA to the filter.