HackMD
Miscellaneous Molecular Biology Methods
By Victuallers - Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=35119980
Measuring concentration and DNA storage
- Use the NanoDrop spec to measure the concentration of DNA. Record this information in your notebook and on the side of the tube. Also note the purity of the sample. Pure DNA should have an A260/A280 ratio ~ 1.8 and an A260/A230 ratio > 1.8
- Store DNA at 4˚C for less than a week or at -20˚C for longer periods.
PCR purification
Many methods exist for the purification of DNA produced in PCR from used Taq and unincorporated primers and dNTPs. Here two methods are described.
Standard QiaQuick PCR purification
If a PCR produces a single band 100 bp to 10 kb, it can be purified using the QIAquick PCR Purification Kit (QIAgen catalog number 28104).
PEG Purification
This method recovers DNA over about 300 bp. Residual PEG should not inhibit most enzymatic reactions. This is the recommended purification method for PCR products used in gateway cloning. It's unclear whether it will be suitable for all applications.
- Add water to your PCR product to bring it to 50 μl total volume.
- Add 150 μl of TE Buffer (pH 8.0; 1X).
- Add 100 μl of 30% PEG-8000, 30 mM MgCl2.
- Vortex to mix thoroughly.
- Immediately centrifuge the tube at 12,000 ×g for 20 minutes.
- Carefully remove and discard the supernatant. The pellet will be clear and nearly invisible. If you remove about ¾ of the solution, then tip the tube, you should see an area at the bottom that looks gelatinous—that’s the pellet containing the DNA. Remove any remaining supernatant, but be careful to keep the pellet.
- Dissolve the pellet in 30 μl of TE Buffer.
