Standard Cloning Protocol (draft: April 19, 2010)
Things to think about before you start…
- If only a couple copies are expected per sample, do 1/3 rxns.
- The A-overhang added during PCR that is necessary for TopoTA cloning will start to
degrade quickly, so gel extraction (and cloning) needs to happen within a few days of
PCR or cloning efficiency will decrease.
PCR:
Amplify samples (possibly with Platinum High Fidelity Taq. Note that the concentration
of the Mg stock and the Taq stock are different).
Gel Extractions:
- Pour a clean 1.5% agarose gel with EtBr using new 1x TAE buffer. Make sure that
there gel rigs are clean and use wide combs with double the number of samples
plus ladder.
- Once the gel is set, fill gel rig with fresh TAE but DO NOT cover the gel. Just fill up
to the edge of the gel so that the current can move from the buffer through the
gel, but so that the wells are dry.
- Load all of each PCR product into wells and add ~ 25µL of buffer to wells that the
ladder will go into followed by the ladder (This just lets the ladder sink to the
bottom and run more cleanly).
- Run for 5 minutes at ~100 V. Then, remove cover and add extra buffer to fill
wells and cover the gel. Run for another 20-30 minutes, depending on fragment
size and gel length.
- Cut bands and put into weighed 1.5mL tubes.
NOTE: The UV light used to visualize the gel eats away at the A-overhang so work
quickly and minimize the time each band spends under the light. You can use foil
to shield the part of the gel not being cut at the time and work one band at a
time. Also remember to use the clean edge of razor blade for each gel slice (to
not cross contaminate), don’t etch the glass of the UV hood, and be careful of
taking too much gel.
- Follow the procedures in the MolBio gel extraction kit – the only exception being
to elute with 30 µL of water rather than 50 µL of Elution Buffer.
- Label with individual, locus, “gel extraction” and date. Freeze until ready to use.
Cloning – Two days:
Morning of Day 1
- Around 10 am, take plates out and put into a 37° C incubating oven with lids ajar
so that condensation will evaporate. Turn the water bath on to 42°C (make sure
it has enough water).
- Check every hour or couple hours to ensure the plates don’t dry out. When the
surface of the agar and the lids are dry, put the lid on (ie, not ajar).
-
Start the cloning reaction around 2pm, assuming you can come in about 18-19
hours later (9am the next morning).
Fill the ice container with ice and put the TopoTA vector directly onto ice. The
salt solution and water should be kept cool, but allowed to thaw – I usually let
them thaw for a few minutes and then put them on ice. Also thaw the gel
extracted PCR product.
1) Label one 2.0mL tube per cloning reaction.
2) Ligation: Working quickly with the p2.5, add to each tube:
0.50 µL water
0.33 µL salt
0.33 µL vector
0.83 µL PCR product
Do not pipette up and down. Instead, add each reagent to the liquid drop at the
bottom of the tube. When adding the PCR product, gently stir the drop and tap
on the counter to collect the liquid at the bottom of the tube.
4) Incubate on the countertop for 5-15 minutes (Note the time).
5) While the ligation is incubation, put the vector, salt, water, and reagents away and
then get the cells from the -80°C freezer on the first floor. These cell tubes need to go
directly onto ice and allowed to thaw on ice. Take one tube per three reactions.
6) At the end of the incubation put the ligation tubes on ice for a minute or two. Spin
down the thawed cells using the mini-centrifuge and pulsing it on just a couple of times.
Using cut, filtered tips with the p100, gently stir the cells and draw up 16 µL and add to
the drop of the first ligation tube gently stirring to mix. DO NOT mix by pipetting.
7) Leave on ice for 5 to 15 minutes.
8) Transformation: Heat shock in a 42°C water bath for 30-35 sec. No more, no less. Do
not disturb the tubes while in the water. Plunge on ice after the heat shock.
9) Leave on ice for ~2minutes and then add 125µl SOC to each (make sure the SOC is
clear, not growing rogue bacteria). Flick to mix.
10) Tape tubes on their sides (make sure caps are on tight) to the backside of a tube
rack and shake at 215-225rpm at 37°C for 1hr15min.
11) Meanwhile, spread 30µL of 20mg/ml X-gal onto the agar plates. Let sit in the oven to
allow residual x-gal to evaporate for at least 1 hr before plating colonies.
12) Plate colonies: Remove tubes from shaking incubator and plates from incubating
oven. Label plates on the agar side and plate ~40-50µL of transformation (this may vary
depending on how efficient your cloning is and how healthy the cells are) and spread
evenly with a flame sterilized rod (make sure it has cooled).
13) Put in oven sitting on lids (agar up) overnight for 16 hours.
14) Check the plates after the 16 hours for colonies… if they are too small you can grow
them longer. Otherwise, proceed or wrap with strip of parafilm and keep in the
refrigerator. The colonies will be good for a few days.
Lysing and BacAmps
1) Pick colonies: Aliquot 25µL TE into strip tubes (8 per cloning reaction). Use the
thermocycler to boil at 95-99°C for 10 minutes and bring down to 4°C.
2) Use lyse directly in a 10 µL PCR with the M13 primers… this is the “BacAmp”.