QIAquick Gel Extraction

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The following method uses the ‘QIAquick Gel Extraction Kit – Centrifuge Processing Kit’ produced by QIAGEN. All centrifugation steps are at 17,900g/13,000rpm in bench centrifuge. Ensure ethanol has been added to the PE buffer and that QG buffer is yellow.

CAUTION – ALWAYS WEAR FACE SHIELD AND BUTTON UP LAB COAT FULLY WHEN WORKING AT THE UV LIGHT BOX

1. Weigh one Eppendorf per each sample to be extracted and label with sample name and Eppendorf mass.
   
   
2. Ensure gel has been stained following Staining DNA Gel protocol. Place ethidium bromide stained gel on the UV light box.
   
   
3. Ensure face shield is on before turning on UV light box at the wall and turning out main room light.
   NOTE: Try to minimise the amount of time your DNA samples are exposed to the UV light.
   
   
4. Use razor blade to cut desired band and place in labelled Eppendorf.
   NOTE: Try to cut away as much excess gel as possible.
   
   
5. Weigh each sample in Eppendorf and calculate mass of sample. Add 3 gel volumes of QG buffer to each sample. e.g. Actual mass of gel sample = 0.2g, gel volume = 200 μl, therefore volume of QG buffer added = 600 μl
   
   
6. Incubate in 50⁰c water bath for 10 mins to dissolve the gel. Vortex every 2-3 minutes.
   NOTE: Keep finger on lid of Eppendorf whilst vortexing.
   
   
7. Check the mixture is yellow. If not, add 10 μl 3M Sodium Acetate and mix.
   
   
8. Add 1 gel volume of isopropanol and mix. If no isopropanol on the bench take a Duran bottle and collect some from the store in the cabinet underneath the fume hood.
   
   
9. Place one QIAquick spin column in a 2ml collection tube for each DNA sample. Label both the spin column and collection tube with sample name.
   
   
10. Pipette sample into centre of spin column and centrifuge for 1 min. Discard flow through and replace spin column in collection tube. The DNA should now be bound to the spin column membrane.
    
    
11. Add 0.5 ml of QG buffer to spin column and centrifuge for 1 min. Discard flow through and replace spin column in collection tube.
    
    
12. Add 0.75 ml of PE buffer to spin column and let stand for 2-5mins. Centrifuge for 1 min. Discard flow through and replace spin column in collection tube.
    
    
13. Centrifuge again for 12 min to remove residual buffer. Discard flow through and collection tube.
    NOTE: Ethanol from the ethanol supplemented PE buffer can inhibit enzymes. To ensure all ethanol is removed from sample, you can stand spin column on cap on the bench to air dry for 5 mins.
    
    
14. Place column in a clean, labelled, 1.5 ml Eppendorf tube.
    
    
15. For Gibson assembly applications a concentrated DNA sample is required, therefore add only 20 μl of EB buffer (elution buffer) to the centre of the spin column membrane. Allow to stand for 4 mins. Centrifuge for 1 min. DNA sample should now be in the flow-through collected in the Eppendorf.
    NOTE: When placing spin columns in Eppendorf tubes into the centrifuge position the caps so that they either point in the opposite direction to the centrifuge rotates or down into the centre of the centrifuge.
    
    
16. To maximise yield repeat steps 14 and 15 to make a second sample of DNA.
    
    
17. Use Nanodrop: Finding DNA Concentration protocol to determine DNA yield.
    NOTE: this will not be an exact measurement as QG buffer interferes with UV/vis readings from the nucleic acid.