Research Protocol: Direct Purification of DNA from PCR Amplifications Using the Wizard PCR Preps DNA Purification System (Promega Corp.) with a Vacuum Manifold
If your chloroplast region-PCR reactions worked (single band of sufficient size and yield), you can proceed to the next procedure of purifying them in preparation for DNA sequencing. Some form of PCR purification is recommended prior to DNA sequencing. DNA sequencing reactions include materials that are very similar to those included in PCR and sequencing requires that these components be in appropriate concentrations. Significant carryover of PCR components disrupts this balance and can cause poor sequencing reaction results.
If you saw small products (< 50 bp) near the bottom of your PCR sample gel run, these are most likely primer artifacts which are generated from the primers interacting with each other or with themselves (aka primer dimers). The following purification procedure not only removes any excess PCR components it also produces very poor yields of products < 75 bp (3% recovery). If performed correctly, products in the expected size range of our targets (around 1000 bp depending on region and species), the expected recovery is approximately 96%.
If other products are co-amplified with your products that would not be removed by this procedure AND are the result of non-specific primer binding, then you can simply use primers that will bind to sites within the amplified sequence. This should allow sequencing of just the desired product. However, if you are dealing with a case of heteroplasmy, multiple alleles or multiple loci (common for nuclear genes), cloning the PCR products into a plasmid might be necessary before DNA sequencing. If these products are of a different size (can be separated on a gel), this kit can be used for gel purification – the PCR products are isolated from excised pieces of the agarose gel).
As our samples tend to amplify with high yield and little background, we cleaning them directly and will be using the same primers for sequencing as were used for PCR.
Before beginning the following procedure, first verify which of your reactions was successful. If both amplifications of a DNA sample worked, you will combine them as part of the purification procedure.
Put on gloves!
Step 1: For each successful amplification from a sampled species, please label one 1.5 ml tubes with the DNA code and region. You can use from 30-300 ul of PCR product in one purification. For specific DNAs where both amplifications were successful, add both to this tube. Since we performed 100 ul reactions and used 5 ul for verification, 95 ul remains (2 x 95 ul = 190 ul).
Step 2: Add 100µL of Direct Purification Buffer. Vortex briefly.
Step 3: Add 1ml of resin (make sure resin is suspended in the stock bottle PRIOR to pipetting. [Warning: wear gloves! The resin mix contains Guanidium thiocyanate, a powerful protein denaturant.]
Step 4: Close the tubes and vortex 3 times over the course of 1 minute (about every 20 seconds).
Step 5: For each sample tube, obtain a Minicolumn and Syringe Barrel. Attach a minicolumn and syringe together via the Luer-Lok (they screw together). [Label these the same as the tube!!] Take each of these to the vacuum manifold (blue “pig”) and insert the Minicolumn end into one of the connections on top of the manifold.
Step 6: Pipet the resin/ DNAs into the appropriate syringe barrel. Turn on vacuum and let the resin be drawn into the minicolumn (to the point that the liquid is removed). If some are drawn through faster than others, turn the top-cock to stop the pull of the vacuum; when all are completed, turn off vacuum. [Under the chemical conditions of the buffer/resin, DNA binds to the resin. The % recovery depends on how well it binds or remains bound during the next step.]
Step 7: To wash the column, add 2ml (2 x 1000 ul) of 80% isopropanol then reactivate vacuum and wait until the isopropanol has exited the Minicolumn. Again, shut stop-cocks as necessary. [This step removes salts and other materials non-specifically bound to the resin.]
Step 8: Dry resin by activating the vacuum for another 30 seconds. Then, remove and transfer Minicolumn to a fresh 1.5ml tube. [Drying the resin is important as residual isopropanol will inhibit DNA sequencing. However, over-drying could lower recovery rates.]
Step 9: Centrifuge Minicolumn at 10,000 rcf (x g) for two minutes. [This step ensures that all isopranol is removed – but does not risk over-drying.]
Step 10: Transfer Minicolumn to a new 1.5ml tube and pipet 50µL of H2O (filter sterilized deionized) and wait a minute. After the waiting is over, centrifuge for 20 seconds at 10,000 rcf.
Step 11: Discard Minicolumn, close tube.
Step 12: Quantify using NanoDrop Spectrophotometer (1 ul). Determine three team members who can 1) collect quantification data (DNA code and quantity of purified) from all team members, 2) organize into a clear table and 3) post this table to the course blog (under the appropriate chloroplast region PCR category). The role of each of these three team members (and the help of any other member of the team) should be noted at the bottom of the blog post.
The desired yield (proposed by the vendor who performs the sequencing) is 10 ul of sample at 50 ng/ul. However, if your yield is less than this, sequence can still be obtained.
Store all DNAs at -20°C (freezer).