John M. S. Bartlett.pdf - Bio-Nica.info

T-Linker Strategy 479
1. Perform PCRs under fairly standard conditions. For example, for cloning the α3 subunit
of the nicotinic receptor, the conditions for amplification were as follows: 3 µL of 10×
PCR buffer, 4.5 µL of 10 mM MgCl 2 , 3 µL of dNTPs (2 mM each), 6 µL of red sucrose,
16.5 µL of H 2 O, 0.75 µL of ha3cpe5 primer (10 µM), 0.75 µL of ha3cpe3 primer (10 µM ),
1 µL of cDNA template, and 0.25 µL of Taq polymerase.
2. Perform PCR using 40 cycles at 94, 52, and 72°C, each for 0.5 min, in a programmable
circulating air oven (ProOven, Integrated Separation Systems).
3. Products were purified by agarose gel electrophoresis and GeneClean (Bio 101; see
Chapter 18).
3.2. Ligation (see Note 4)
1. Assemble ligation reactions as follows: 2 µL of purified PCR product, 7.5 µL of H 2 O,
2 µL of 10× ligase buffer, 1 µL of (0.5 mM final) 10 mM ATP (if not in buffer), 2.5%
(5% final) 40% PEG 8000 (optional), 2 µL of T-linkers (5 µM in 500 mM NaCl), and
1 µL of ligase (1 U/µL dilution).
2. Incubate at room temperature for 20 min to overnight.
3.3. Reamplification with T-Linker Primer
1. After ligation of the T-linker, reamplfy the products as follows: 5 µL of 10× PCR buffer, 7.5
µL of 10 mM MgCl 2 , 5 µL of dNTPs, 10 µL of Red sucrose, 16.5 µL of H2O, 5 µL of TL-A
(or HisTL-A) primer (10 µM ), ligation mixture, and 0.35 µL of Taq polymerase.
2. Cover the sample with mineral oil and amplify with 40 two-step cycles of 94°C for
0.5 min and 72°C for 2.5 min. (see Note 5).
3.4. Alternative Method: One-Tube Ligation/Reamplification
The whole T-linker reaction can be set up in one tube to make a “kit-like” product.
This is done using a meltable barrier, as for hot-start PCR (we use AmpliGrease; ref. 7).
The “top mix” contains the ligation reaction, to which the PCR-amplified band is
added. After a suitable incubation to allow ligation, the reaction is heated to melt the
barrier, and the second PCR is begun:
1. Set up a 100-µL PCR mix (see Subheading 3.1.) in a tube. Add only TL-A (or HisTL-A)
as a primer, and no template.
2. Dispense approx 35 µL of petroleum jelly (AmpliGrease) onto the side of the tube with
a syringe.
3. Heat the tube so that the grease melts to cover the bottom mix.
4. Allow to cool so that the grease resolidifies.
5. Add approx 30 µL of mineral oil on top of the grease.
6. Add 4 µL of ligation mix through the oil so the droplet rests above the grease barrier. The
ligation mix is made as a master mix containing the following: 2 µL of T-linkers (5 µM each
in 50 mM NaCl), 2 µL of 10× ligase buffer, 1 µL of 10 mM ATP (if not in buffer), 0.5 µL
of ligase (8 U/µL), and 10.5 µL of H 2 O.
7. Add 1 µL of the PCR product to be cloned into the droplet of top ligation mix.
8. Use the following reaction conditions: 25°C for 1 h (ligation); 94°C for 15 s, 55°C for 15 s,
and 72°C for 45 s for 30 amplification cycles (see Note 6).
3.5. Digestion and Cloning
The reamplified PCR product now has the restriction sites from the T-linker at its
ends, and may be cloned using standard procedures. The protocol we usually use is
as follows: