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

508 Ravassard et al.
4. Wash twice with 1× PCR buffer, 5% SDS, for 5 min at room temperature.
5. Wash with 1× PCR buffer until SDS is completely removed. To do this, change the
microtube after every wash.
3.7.3. PCR Amplification of the Captured cDNA
1. Transfer one fourth of the beads with the captured cDNA into a PCR tube. Make sure
that all traces of SDS are removed.
2. Perform PCR amplification with A5′_3 and A3′_3. Use the same protocol as described
in Subheading 3.6.
3.8. Blunt-End Cloning of PCR Products
For blunt-end cloning, the 3′ overhanging extremities of the PCR product are
removed with T4 DNA Polymerase (3′–5′ exonuclease activity). Oligonucleotides
usually have 5′ hydroxyl ends. To allow ligation of the PCR product those extremities
have to be phosphorylated by T4 Polynucleotide kinase (T4 PNK).
1. At the end of the amplification reaction, add to the PCR mixture 1 µL of T4 DNA
polymerase (4 U/µL). Incubate for 20 min at 16°C. Do not allow the temperature to
rise above 16°C.
2. Load the PCR product on a preparative agarose gel.
3. Cut the desired bands and purify the DNA with the QIAEX II purification kit. Follow
the supplier’s recommendations.
4. Elute DNA from the silica matrix with 10 µL of ddH 2 O.
5. Add 1.5 µL of 10× T4 PNK buffer, 1 µL of 3 mM ATP, 1.5 µL of ddH 2 O, and 1 µL of
T4 PNK (5 U/µL).
6. Incubate at 37°C for 30 min.
7. Heat inactivate the enzyme at 75°C for 20 min.
8. The PCR product is ready for ligation. Use the same buffer as the phosphorylation reaction,
a dephosphorylated blunt-end vector (e.g., pUC19 SmaI), and a final concentration of
ATP of 0.8 mM.
9. Transform by electroporation and plate on the appropriate selection medium.
3.9. Direct PCR on Colonies (see Note 8)
1. Pick a colony with an inoculating needle.
2. Touch the bottom of a 0.5-mL microtube (or a well in a microtiter plate) with the needle.
3. Inoculate with the same needle, 3 mL of liquid bacterial growth medium in a 15-mL tube,
and incubate at 37°C for 18 h.
4. Repeat steps 1 to 3 for all the colonies to be analyzed.
5. Prepare the PCR mixture on ice as follows. The volumes given are sufficient for one
reaction: 2.5 µL of 10× PCR buffer, 2 µL of 2.5 mM dNTP, 2 µL of 50 mM MgCl 2 , 2 µL
of primer 1 (50 ng/µL), 1 µL of primer 2 (50 ng/µL), 0.1 U of Taq DNA polymerase,
and ddH 2 O to 25 µL.
6. Distribute the PCR mixture into every tube on ice and add 100 µL of mineral oil.
7. Place the tubes or the microtiter plate in the thermal cycler and run the following program:
3 min denaturation at 93°C; 35 cycles of 94°C for 30 s, 55°C for 45 s, and 72°C for
1 min/kb.
8. Analyze the PCR products on an agarose gel.
9. Prepare plasmid DNA of the positive clones from the cultures (prepared in step 3). Use
this plasmid DNA for sequencing.