1. Amplifying from a plasmid or isolated sample of DNA
You have a tube of linear or plasmid DNA like that from the registry directly and don’t want to wait for the the transformation and miniprep. (note: you should go through the time-intensive transformation in parallel regardless).
In this case, you need first to know the concentration of your sample. If you don’t know it or it was not provided, you can learn the concentration for your sample by using the nanodrop machine located in room 347. It depends on the size of your template, but as a general rule, you need on the order of 25-50 ng template minimum for a successful PCR, so adjust the volume of your template in your PCR accordingly.
2. Colony PCR
You can also amplify plasmid or genomic DNA straight from live cultures of organisms containing your desired sequence. You will usually have cultures in one of two forms: either in liquid culture, or spread on an agar plate. If you are amplifying from liquid culture, grow it up as much as you can and add 1μL of the culture to the PCR mix. If you're amplifying from the plate, there is no need to add a volume; instead, simply take a pipette with a pipette tip from the green box, gently touch the pipette tip to the desired colony on the plate (try to take as little from the plate as possible; agar can screw up PCRs), and then insert your pipette tip into the
PCR mixture and pipette up and down to mix.
Q5 Polymerase
Q5 is a fast, high-fidelity polymerase that even beats Phusion. Unlike Taq, Q5 produces blunt-end amplicons. It’s also very expensive so treat it carefully.
25 μL recipe: 5 μL 5x Q5 buffer
0.5 μL 10mM dNTPS
1.25 μL forward primer (10μM dilution)
1.25 μL reverse primer (10μM dilution)
Template DNA (a couple nanograms worth)
qH2O to 24.75 μL
0.25 μL Q5 enzyme (add last)
The 50uL recipe (when you needs lots of product) is simply double.
Thermocycler Conditions
Taq polymerase (GoTaq Green) Initial Denature: 95°C 2 min
The official Platinum Blue protocol calls for
94°C for 3 min, although I have never done it that way. Either will work, I am sure.
Denature: 94°C 15-30 secs
Use a shorter time if the amplicon is a relatively short segment of DNA, and a longer time if it is a relatively long piece of DNA.
Annealing X°C 15-30 secs
This is the most crucial step of the thermocycle! Your annealing temperature will be determined by the melting temperature of your primers. As a general rule, your annealing temperature should be about 5° lower than the lowest melting temperature of your primer pair. Additionally, if you are trying to add tails to you amplicon (e.g. you are trying to add restriction sites to the ends of your DNA template), you may need to drop the annealing temperature down even more. I have had primers with melting temperatures above 65° that needed to be annealed at 42°.
Additionally, if a primer may be difficult to anneal to the template, you can increase the annealing time for better results.
Extension 72° X seconds. Taq extension runs at 1kb per minute. Therefore, allow the extension step enough time to fully copy your entire amplicon. Repeat steps 2-4 32X. Final Extension 72°C 5 min. Hold 4°C forever.
Q5
Initial Denature at 98°C for 30 sec. Denature at 98°C for 10 sec. Annealing at X°C for 15-30 sec. Use the NEB calculator (linked here). Extension at 72°C for X seconds. Q5 is much faster than Taq, and requires 20-30 sec per kb. Go to step two 25-35X. Final extension at 72°C for 2 min. Hold 10°C forever (zero minutes=forever)
The standard protocols for various polymerases can be found at the respective company sites: GoTaq and Q5.
PCR Cleanup (using Wizard SV Gel and PCR Purification System)
Gel Extraction:
Following electrophoresis, excise DNA band from gel and place gel slice in a 1.5ml microcentrifuge tube. Trim the slice of parts that don’t contain DNA. Weigh gel slice (by weighing the tube containing the slice and subtracting the mass of the empty tube). Add 10μl Membrane Binding Solution per 10 mg of gel slice. Vortex and incubate at 50–65°C until gel slice is completely dissolved (usually 10-15 minutes).
PCR Amplifications:
Add an equal volume of Membrane Binding Solution to the PCR amplification.
Binding of DNA
Insert SV Minicolumn into Collection Tube. Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate at room temperature for 1 minute. Centrifuge at max speed for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube. If you are worried about the final concentration of your purified product, you can repeat this step to maximize the amount of DNA bound to the filter.
Washing Add 700μl Membrane Wash Solution (ethanol added). Centrifuge at max speed for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
Repeat Step 4 with 500μl Membrane Wash Solution. Centrifuge at max speed for 5 minutes. Empty the Collection Tube and re-centrifuge the column assembly for 1 minute with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.
Elution
Carefully transfer Minicolumn to a clean 1.5ml microcentrifuge tube. Add 30-50 μL of Nuclease-Free Water to the center of the minicolumn. Incubate at room temperature for 1 minute. Centrifuge at max speed for 1 minute. By adding less water, like 30 μl, you will increase the concentration but decrease the total amount of product. On the flipside, if you want to maximize product, you can maximize elution volume so long as you don’t care about concentration.
Note: you can also increase yield by warming the elution water before hand. I usually warm it to 40°C with good results.
Discard Minicolumn and take sample to nanodrop (see 'Nanodrop', below)
Store DNA at –20°C.
The standard protocols for the SV Wizard Gel and PCR purification kit can be
found here.
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