1. Electrophorese PCR product using a 1.5% TAE gel (50 µl for 7 µl sequencing template).
While the gel is running, tare one 1.5 ml microfuge tube for every sephaglas product and record the tared weight on the cleanup record.
2.Cut out desired product from gel. Visualize the bands under long-wave UV light in a darkroom and cut bands out of gel with a clean scalpel. Once the agarose plug is out of the gel, break it into smaller pieces and place it in the appropriate tared microfuge tube.
3. Determine the mass of the agarose/DNA plug.
4.Use the attached chart to determine the amount of gel solubilizer needed in each tube
(1.5 X gel solubilizer per mg of agarose) and add to each tube.
5. Vortex tubes vigorously and incubate at 60 C for 5-10 minutes or until gel is completely solubilized. Make sure that the agarose plug is completely immersed in the solubilizer solution during the incubation step.
6.When the agarose is completely solubilized, add 15 µl Sephaglas BP (make sure the sephaglas is a uniform suspension before using) to each tube, vortex gently and incubate for 5 minutes at room temperature, vortexing every minute.
7.Pulse spin for 30 seconds and remove the supernatant, making sure not to disturb the sephaglas pellet. Do this by inverting tube and then drying with the corner of a kimwipe.
8. Spin again for 30 seconds and remove residual liquid with a kimwipe without disturbing the sephaglas pellet.
9.Add 160 µl Wash buffer (make sure EtOH has been added to the wash buffer solution) to the pellet and pipette up and down to resuspend the pellet. Pulse spin for 30 seconds and pour off the supernatant without disturbing the pellet. Repeat this step two more times (total of three washes.)
10.Tap the tube to disperse the pellet and let dry at room temperature for 10-60 minutes. Do not dry under vacuum.
11.Add desired quantity of elution buffer (20-50 µl; appx. 7 µl elution buffer for every 50 µl initial PCR product but you can use more elution buffer without much problem), vortex gently to resuspend pellet. Incubate at room temperature for about 5 minutes with periodic agitation.
12. Spin at high speed for one minute in a microcentrifuge and place the supernatant into a clean microfuge tube, making sure to avoid the sephaglas pellet. Store at -20 C until needed.
13.Run 1 or 2 µl clean product on gel to estimate amount of template and store labelled product at -20C until sequenced.
|gel weight (grams)||Solubilizer vol. (µl)|
|> 0.50||split into 2 tubes|
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