Buffer QG Gel Purification

 

While this protocol is apparently robust, Buffer QG contains Guanidine Thiocyanate. This is potentially the most hazardous chemical in the entire discipline, approach this buffer and this protocol with extreme caution.

Equipment & Consumables:

Protocol:

  1. Put on gloves + safety glasses. QG contains guanidine which is extremely toxic

  2. If your weight of agarose is less than 100 mg (=100 µl), then make it up to 100 µl with TE. The volume you now have will be called “1 volume”. Then add “3.5 volumes” of QG buffer to your gel slice. eg. if you have 100 mg of agarose, add 350 µl of QG.

  3. Slice up or mash the agarose in an Eppi (<300 mg agarose) or a McCartney bottle (>300 mg). This will speed dissolution. Melt the agarose with heating (60°C for 5 min is usually enough, mix occasionally), then when it’s all dissolved, allow to cool to room temp.

  4. Add “1.5 volumes” of isopropanol to the mixture. e.g. for 100 mg agarose, use 150 µl isopropanol.

  5. Place a silica-based spin column (e.g. ‘Econospin’) into its 2 ml catch tube (if it isn’t already set up that way). Load up to 750 µl of the DNA / QG / isopropanol mixture onto the column. Spin at ~10,000 g for 30 sec. Discard the flow-through into culture waste.

  6. If you still have more DNA /QG / isopropanol mixture left, repeat the previous step until all of the mixture has been put thru the column. The columns will hold a total of ~10 µg DNA, which is a lot!

  7. Add 750 ml of buffer PE to the column, spin ~10,000 g for 30 sec, discard flow-through.  

  8. Repeat step 6.

  9. Spin again for 30 sec to remove all traces of PE from the column. Discard both the flow-through and catch tube, and transfer the spin column onto a clean Kimwipe. Leave the column lid open. Transfer Kimwipe to 60°C oven, and allow to dry for 10 min.

  10. Transfer spin column to a sterile 1.5 ml Eppi tube, and add 20-50 ml* of EB buffer (5 mM Tris, pH 8) to the centre of the spin column – ie on the membrane, not the walls of tube. Allow to sit for 2 min. Spin at ~10,000 g for 1 min, retain Eppi tube with DNA solution in EB, discard spin column. 

    • *Usually we want high concentration rather than high yield, so use 20 µl. If max. yield is important, or if you have lots of DNA, use 50 µl. Note that you lose approx 5 ml EB during the procedure.  

Acknowledgements: