Gel extractions can be a low yield affair, but overnight incubation over a spin column can increase yields. This protocol details a modified QIAquick Gel Extraction protocol.

QIAquick Gel Extraction Kit 

1. Cut the desired band out of an agarose gel using a scalpel. 

2. Weigh the slice in a snap cap tube and add 3 volumes of QG buffer per volume of gel, assuming the gel density is roughly 1µl/mg. This can be whatever gel dissolving buffer (with chaotropic salts and guanidine thiocyanate) you typically use.

3. Add 1 volume of isopropanol to the tube and vortex.

4. Place a QIAquick spin column in a collection tube. Add the sample to the column and incubate over column overnight at room temp. 

5. Centrifuge spin column for 1 minute at 13,000 RPM. 

6. Discard flow through. Repeat step 5 if the sample volume was greater than 800 µl.

7. Add 750 µl of buffer PE to column and centrifuge for 1 minute at 13,000 RPM. Discard flow through.

8. Centrifuge for an additional 1 minute to remove all wash buffer.

9. Place column in collection tube and add 50 µl EB buffer or water directly to the silica portion of the column. Incubate up to 4 minutes then centrifuge the column for 1 minute to elute DNA.

Analyze DNA using a standard Nanodrop/general spectrophotometer protocol for 260/280 absorbance ratios. Store all samples in a -20 oC freezer.

Higher Yield Gel Extractions

PCR can be performed directly from a scraped cell colony, using an extended denaturing step to cause lysis of the cells. This is a nice time saving protocol for PCRing targets directly from antibiotic selective plate media. 

Colony PCR with Patch Plating
1. If subculturing via patch plate, divide a warm LB-Amp plate into a grid (commonly 3x3), labelling each square on the back of the plate.

2. Using a sterile wooden stick, aseptically pick up a transformed colony and smear some of the colony in a PCR tube. 

3. If subculturing via patch plate, use the same stick to patch it on one of the squares. 

4. Repeat steps 2 and 3 for multiple colonies.

5. Place patch plate in 37oC incubator. Move to a 4oC fridge the next day.

6. Add reagents to the PCR tubes as described in table 1. The reagent volumes can be modified to fit whatever your typical PCR setup looks like.

7. Run the PCR cycles as detailed in table 2.

8. The resultant PCR products can be run through a gel for gel extraction or cleaned with a PCR cleanup kit.

Figure 1 displays the results of the colony PCR using all 6 patched colonies. Lanes 1 - 6 contain the colony PCR products of 6 transformed colonies. A 1 kb ladder was loaded into lane 7. Lanes 8 and 9 were PCR products of untransformed control colonies. 


PCR

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