Patch Plate & Colony PCR

(Junction PCR & Spanning PCR)

So there are colonies on your ligation/assembly plate and nothing suspicious on any of your control plates! Well done! Now to screen them and organise them nicely on a fresh plate and run some very specific PCRs.

Flanking Primers.png

Nick Breaks Down Junction PCR:

“Left Junction PCR”
The PCR using primers F1-R1 is the best starting point for screening– this is the ‘left junction’ PCR. If you get a product of the expected size in this PCR, this is a good indication that you have the desired recombinants.

“Right Junction PCR”
The PCR using primers F2-R2 (right junction PCR) can then be done on clones that are positive in the F1-R1 PCR. If the clones are also positive in this PCR, this is further evidence that they are the right thing.

“Spanning PCR”
The PCR  using primers F1-R2 is known as a ‘spanning’ PCR or ‘insert spanning’ PCR – this will amplify the whole insert region. This PCR can be used to determine if the clones have an insert of the expected size, but note that this is not an all or nothing PCR, unlike the junction PCRs. In the spanning PCR, the clones will give a small product if they have no insert, and a large product if they have an insert. This PCR is a bit ambiguous though, since if you have a mixed population of cells in your template DNA, you may see only the small product (this is amplified preferentially), even if you do have some positive clones in there too. The spanning PCR is most useful in preparation for sequencing the insert DNA region, rather than for initial clone screening.

The positioning of the screening primers is important for later steps (sequencing). Make sure that the primers F1, R1, F2, R2 are at least 100 bp away from the ligation join – this is because the first ~50 bp of sequence read are usually ‘junk’. Also ensure that these primers are not *too* far from the ligation join, otherwise you are amplifying (and later sequencing) stuff that you don’t need to, and it makes the PCR take longer to thermocycle. Placing the primers about 150-200 bp from the ligation join is good, which gives an overall PCR product size of 300-400 bp.

Equipment and Consumables:

Initial Patching & PCR Screening

Patch Plate.png

  1. Choose 20 well-isolated colonies from your transformation plates. Circle these and number them on the base of the plate.

  2. Using an agar plate containing the appropriate antibiotic, divide a fresh LB-Agar-Antibiotic plate into patches, using a square grid (5 x 5). Label twenty of the squares with numbers 1 to 20. (see diagram above).

  3. Label three PCR strip tubes with numbers 1- 20, leaving three blanks (one in each strip) as negative controls. (so your first strip should be clones 1-7, the next one should be clones 8-14, and the last one should be clones 15-20)

  4. Prepare a PCR master mix with the appropriate left junction PCR primers, enough for 21 (n+1) PCRs, aliquot 25 µl amounts into the labelled PCR strip tubes. If there are less than 20 colonies to screen, adjust your numbers appropriately. Taq polymerase is good for this job - it is very robust to ‘mess’ in the PCR, but other polymerases can also be used.

    • 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) Taq DNA Polymerase (0.05 U / µl)

      • Cells stirred in from pipette tip

    • EXAMPLE 8 REACTION MASTER MIX
      Rxn Volumes x 25 (n+1)

      • 500 µl Sterile dH2O

      • 62.5 µl 10x buffer (1 x)

      • 12.5 µl 10 mM dNTPs (200 mM)  

      • 6.25 µl 50 µM Primer #1  (0.5 µM)

      • 6.25 µl 50 µM Primer #2  (0.5 µM)

      • 6.25 µl (5 U/µl) Taq DNA Polymerase (0.05 U / µl)

      • Cells stirred in from pipette tip

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

  6. Using a white pipette tip, pick up some growth from the colony of your first clone of interest. Make sure you can see some cells on the tip, but you don’t need a lot (approx 1 mm3 = 1 µl).

  7. Dip the tip in and out of the mix in the first PCR tube five times. You don’t need to dislodge the whole chunk of growth, enough cells will fall into the mix to give you a product, if the gene of interest is there. This step is why it is called a colony PCR.

  8. With the same tip, transfer the remaining growth onto the first patch on your agar plate, by scratching the tip across the surface of the agar a few times (e.g. make a set of three closely-spaced parallel lines – see diagram). Stay well clear of the borders of each square, we don’t want the clones to touch each other when they grow up. Then discard the tip.    

  9. Repeat steps 3 and 4 with the remaining (n) clones, being very careful to keep track of which colony, which tube, and which patch you are up to (this is why labelling before you start is important!) 

  10. 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. Incubate the patch plate overnight at 37°C.

  11. Return all reagents to the freezer.

Thermal Cycler Set-up

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
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

    • Rule of Thumb:

      X = (Average Tm of Primer #1 and Primer #2) - 5

      • Good starting point, but only works about half the time in my experience

      • If you want a second opinion on the Tm calculated by Snapgene/Benchling try the NEB Tm Calculator. It will also recommend a value for your Annealing Temperature (X), which I have found to be reliable.

    • Other Annealing Temperature calculators:

    • X will have a range of values, with the centre of that range likely being ideal. ie. you may get some product at 52°C and 56°C, you’ll likely get a better yield at 54°C.

  • 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)

    • Start by knowing the exact distance between the 5’ ends of Primer #1 and Primer #2 on your template strand. Next, check the speed of your polymerase in bp/min.

    • E.G. A 1,200 bp fragment amplified by Taq polymerase (2000bp/min) = 60 seconds

    • E.G. A 1,200 bp fragment amplified by Pfu Polymerase (500bp/min) = 2 minutes 30 seconds

    • Always round up to the nearest 30 seconds - Polymerase speed is often measured in minutes and as a result can be a bit inaccurate. Generally adding a bit of excess time will not negatively affect the reaction. On the other hand, underestimating (Y) will cause problems.

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

*Opportunity to sleep while the Thermocycler runs*

Agarose Electrophoresis:

After thermocycling is completed, run your PCR reactions out on an agarose gel to check their size and yield by comparing the size of the bands to a DNA ladder. Typically, we would load 5 µl of PCR mixture. Colony PCRs will typically have uneven or ‘wavy’ bands due to the impedance of DNA migration through the gel by cellular debris. If you don’t have a 20 well agarose gel (that’s a massive gel) consider running 2-3 gels. If you’re lucky, you’ll have a few positive clones early on and it won’t be depressing. Alternatively, accept the limited bandwidth of your lab and produce patch plates with 10-11 colones.

  1. Write down a “Load Order” of samples. Remember to leave the first (and possibly last) lane free for your DNA ladder.

  2. Decide what agarose concentration, running buffer and staining method are appropriate to maximise band resolution.

  3. Set up your gel-casting tray, ensure the well Comb is not touching the sides, or bottom of the Gel Tray.

  4. Weigh out the appropriate amount of agarose powder (NOT AGAR!) for the correct % agarose gel that you calculated in step 2 into a small, clean, dry conical flask, then pour in the appropriate volume of TBE or TAE buffer. Stir gently, ensuring no agarose is stuck on the edges, then put in microwave, and heat on high for 1 minute.

  5. Put on heat resistant gloves, then take out to examine whether all the agarose is dissolved. If the solution is still cloudy, replace for another 30 sec on high power, and repeat examination and heating until all dissolved.

    • Be very cautious of overboiling! The surface tension of agarose can lead to violent eruptions of molten agarose. Heat resistant gloves are a necessity, don’t put your face near the conical flask opening

  6. Pre-Stain: While the agarose is nice and fluid, add an appropriate amount of your intercalating staining dye to the gel and gently swirl the conical flask to mix it thoroughly.

  7. (if using plastic end-formers): using a p-1000 pipette, pipette a thin line of agarose along the bottom edge of the end-formers, where they meet the base of the casting tray – the aim is to seal this crack through which agarose can potentially leak out. Allow 30 sec or so for this agarose seal to set.

  8. Pour the molten agarose into the casting tray. Use a single smooth motion, don’t stop and start. Stop when the agarose is 3/4 of the height of the ‘teeth’ on the well-forming comb. Or alternatively, stop when you judge the wells are deep enough to hold the amount of sample that you need to load.

  9. Allow the gel to set, this takes about 15 minutes. You can use this time to arrange your samples in the ice box according to your load order, quickly defrosting the DNA ladder in your hands before leaving it on ice.

  10. When gel is set, pour a little of the appropriate buffer (TBE or TAE) over the top, then carefully pull out the well comb (straight up, don’t yank them side to side or forwards and back). The reason for the buffer is to stop the wells collapsing on themselves (this can happen with thin wells at lower % agarose). Then pull out the end-formers or take off the masking tape.

  11. Fill the gel tank up with the appropriate buffer so that it fills the reservoirs on both sides of the gel, and so that it *just* covers the gel (by 1-2 mm).

  12. Prepare a row of spots of loading buffer on a Parafilm strip, of appropriate volume and number. The NEB Purple loading buffers are 6x conc, so e.g. you will need 2 µl spots if you are loading 10 µl samples.

    • You can mix the dye directly into your samples if this gel is your final step and you do not wish to use them again.

  13. 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.

    • The PCR samples will be “cloudy” at the bottom due to cellular debris. Try to take your sample off the top part of the mixture in the PCR tube.

  14. 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 the buffer system and gel size (see later section for more detail). Check that you have current (non-zero milliamps), and gas bubbles at the electrodes (in clear tanks). If not, check all the wire connections. Running the gels at lower voltages generally gives better resolution of bands (maybe! depends also on other factors). 

  15. 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.

  16. 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.

  17. 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.

Secondary PCR Screening

  1. Examine the patch plates prepared above Choose up to seven of the clones that gave a positive PCR in the left junction screen (at least three is recommended).

  2. Set up a PCR master mix using the right junction primer set. The protocols are identical, except swap out the primer set.

  3. Screen the putative positive clones as described above, by colony PCR. Again, be careful to label everything before you start. Pay close attention to which clone patch is going into which PCR tube.

  4. After thermocycling, run out the PCRs on agarose, and note which are positive for the right junction. Choose one of these for a plasmid prep; use this clone to inoculate 100 ml LB-antibiotic broth (see other protocol for details of plasmid prep).

  5. Keep the patch plate! (wrap in parafilm, and store in cold room) – you may need to come back to this plate to plasmid prep a different clone if there are problems with the first one.

  6. Move on to the Restriction Digest Analysis Protocol

Acknowledgements: