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Gel extraction and purification of the PCR products ran on a gel, and of the digested backbones
Protocol: Gel Extraction
A gel extraction is used to select a fragment of DNA of a specific length
out of a solution composed of different fragments (ideally the difference
in length between the wanted fragment and the closest-sized fragment should
be more than 200bp). These fragments are often obtained after a
digestion.
The yeild for this procedure is typically very poor so a large amount of starting material, digested DNA in this case is required. We typically used 4 micrograms. The digestion products are loaded on a gel. Lanes on both sides of the one to be extracted should be empty to make cutting easier and avoid contamination with other fragments.
The gel should be run long enough for the bands to be spread out. This is particularly important if the fragment of interest is around the same length as other expected digestion products. UV light is necesary to observe the bands on the gel but exposure time should be minimized to avoid DNA damage. The fragment of interest is then excise and put in an Eppendorf (consider using a 2ml one).
To extract the DNA from the agarose we used Macherey-Nagel's "Nucleospin® Gel and PCR clean-up" kit. Its manual can be found here: [http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf Gel and PCR clean-up Manual]
Tips
- Cut away as much Agar as possible without slicing into the DNA. Excess agar will require more solvent to dissolve and will result in a poorer yeild upon elution.
- Minimize the DNA's exposure to the UV-light. UV will damage DNA and have negative effects on any subsequent reactions (for example, ligations can be 10'000x less effective when DNA has been exposed to too much UV light [http://openwetware.org/wiki/DNA_Ligation]
The backbones prepared for ligation and the PCR products were extracted from the gel and purified.
Nanodrop check
Protocol: DNA Concentration Measurement
- Take a 6 µl aliquote of the DNA and put back the main DNA tube in the fridge.
- Go to the room by the E.Coli lab (LBTM, not on Friday morning!) with:
- The 6 µl aliquote
- A 10 µl pipet
- Optionally, the buffer you used for DNA elution (there might be some next to the machine).
- The machine is the NanoDrop Spectrophotometer.
- On the computer, click on "Nucleic Acid".
- Put a 2 µl drop of (nuclease-free) water on the machine's tip as you are asked to and measure.
- Clean tips (both sides) with a quarter of tissue.
- Add 2 µl of the buffer you use and click on "Blank".
- Clean tips (both sides).
- Add 2 µl of your DNA sample and click "Measure".
- Clean tips (both sides) with a tissue.
- Take 2 measurements per sample (for averaging).
- Print the report when you are done
- Click on exit.
The important numbers are:
- 260/280 ratio, must be > 1.8
- 260/230 ratio, must be > 2 (too big, > 2.5? , might mean too much salts)
- Of course the DNA concentration.
The Nanodrop showed very low values for the gel extraction, the loss of DNA is huge.
