Gradient PCR

This protocol is excellent for determining the optimal binding temperature for a set of primers, however it requires a special kind of thermal cycler. Check out this page for more general information on the Polymerase Chain Reaction.

Equipment and Consumables:

Gradient PCR Setup Protocol:

master mix preparation

EXAMPLE 8 REACTION MASTER MIX
(Rxn Volumes x 9 (#Reactions + 1 for pipetting tolerance)

  • 180 µl Sterile dH2O

  • 22.5 µl 10x buffer (1 x)

  • 4.5 µl 10 mM dNTPs (200 mM)  

  • 2.25 µl 50 µM Primer #1  (0.5 µM)

  • 2.25 µl 50 µM Primer #2  (0.5 µM)

  • 2.25 µl (5 U/µl) DNA Polymerase (0.05 U / µl)

  • DNA template:  8 x 1 µl

EXAMPLE 25 µl REACTION MIX (Final Conc.)
(Amount pipetted in each PCR tube after MM)

  • 20 µl Sterile dH2O

  • 2.5 µl 10x buffer (1 x)

  • 0.5 µl 10 mM dNTPs (200 mM)  

  • 0.25 µl 50 µM Primer #1  (0.5 µM)

  • 0.25 µl 50 µM Primer #2  (0.5 µM)

  • 0.25 µl (5 U/µl) DNA Polymerase (0.05 U / µl)

  • DNA template: 1 µl

reaction set-up

  1. Calculate how many PCRs you need altogether, and thus the total volume of master mix required. It’s important to make more than you need (~10% more), as pipetting errors will always change the expected volumes a little bit. As a general rule, calculate (number of reactions you need + 1) and then multiply the volumes in the above left hand column to get the required volumes for your master mix.

    • To maximise the effectiveness of your gradient, set up enough reactions to use every well in a single row of the thermal cycler. This is generally 12, but sometimes can be 8 in smaller machines.

  2. Label your strip tubes (1, 2, 3, 4, …, n)

  3. Retrieve the PCR reagents from the -20°C freezer and thaw them (except polymerase, this remains liquid even at -20°C due to glycerol in buffer).

  4. Prepare the master mix in a sterile Eppi tube. Since the variable in this experiment is the annealing temperature, every tube should contain the same reagents.

  5. Aliquot out the master mix between all of the PCR tubes, putting 25 µl in each tube.

  6. Put lids on tubes, ensure they are snapped on tight, place immediately in thermocycler. Double check your program parameters before starting. See below for detailed thermocycling instructions.

  7. Return all reagents to the freezer.

Thermal Cycler Setup

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Initial denaturation:  95°C, 5 min 
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Denaturation:          95°C, 30 sec, 25-35 cycles

Annealing:               X°C, 30 sec, 25-35 cycles

Extension:               72°C, Y min, 25-35 cycles
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Final extension:      72°C, 10 min

Hold:                       15°C      
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

The letters that are underlined and in bold indicate variables that need to be optimised for every individual PCR.

  • X = Annealing Temperature which primers will bind

  • Y = Extension Time that the polymerase will need in order to amplify your segment.

    Y (minutes/seconds) = Length of Target Gene (bp) ÷ Speed of Polymerase (bp/min)

  • Occasionally Extension/Final Extension temperature might change between 68-72°C. It might be worth checking the optimal temperature for your specific polymerase.

Agarose Electrophoresis Protocol:

After thermocycling is completed, you can see the result if you run your PCR reactions out on an agarose gel. You can set up and pour the gel while the reaction runs, or do it the following day if it’s getting late. Typically, we would load 5 µl of PCR mixture. This should give a very strong band if the PCR has been successful. You must keep the amount added constant as the relative brightness of the bands will allow you to quantify the success of the reaction.

  1. Set up and pour a 1% Agarose gel appropriate to your chamber e.g. 0.5 g Agarose in 50 ml TAE buffer. Include ~5µl of intercalating dye if you plan on prestaining.

    • Pre-staining works well but post-staining is ideal.

  2. Remove the gel comb and set up the gel in the bath so that the buffer just covers the top of the gel by 1-2 mm.

  3. Prepare a row of 2 µl spots of loading buffer on a Parafilm strip, of appropriate volume and number. Mix your DNA ladder with first blue spot by pipetting up and down (avoiding any bubbles) and then load the entire volume of the spot into the first well. Steady the pipette tip with the finger of your non-pipetting hand to ensure accurate dispensing. The tip needs to be just inside the well, don’t push it all the way down in the well. Change tips, mix up the next sample with blue dye, and load again. Repeat for all samples according to your written load order. If you make a loading mistake, take note of this on the load order.

    • Alternatively you can add 5 µl to your 25 µl PCR tube and mix. This will make it useless for anything downstream and is a bit of a waste of loading dye - but is a lot less tedious.

  4. Put the lid on the gel box, check that the terminals are connected correctly (negative terminal should be closest to the wells, positive terminal is far from the wells, ie. Run towards Red). Run the gel at ~50 volts up to ~300 V depending on what you’ve found to be most successful with your current set up.

  5. Stop the gel when the fast-running blue dye (bromophenol blue) (= usually the only blue dye) is near the end of the gel (this may take 10-120 min depending on gel size and voltage). You may need to run longer to get good separation of large products (>5 kb). Run for a shorter time for small products (<500 bp), or they may run off gel.

    • Pre-Stain: If you added intercalating dye to the gel before pouring, it is now ready to image on the transilluminator. Follow machine specific instructions and take an image while illuminating at a wavelength that corresponds to the emission spectrum of your chosen dye. Otherwise, follow post-stain procedure.

    • Post-Stain: Add gel to 100 ml of post-stain solution to plastic tray, gently slip your gel into the solution and then cover the tray with foil. Stain gel on rocking platform or orbital shaker (gentle shaking!) for 30-60 min or overnight. This staining solution can be reused a few times. Keep the post-stain solution covered in foil and keep in a closed plastic box so it doesn’t evaporate.

Example Gel: Here you can clearly see that the lowest temperatures in the gradient were the most successful/produced the most product;

Gel was run at 200V and thus has a ‘smile’. All products are actually of identical size, the curve is a relic of impatient electrophoresis.

Gel was run at 200V and thus has a ‘smile’. All products are actually of identical size, the curve is a relic of impatient electrophoresis.