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

T4 DNA Polymerase 471
2. Amplification conditions largely depend on the specific applications. However, a general
cycling profile can be used in most experiments: 94°C for 7 min (initial denaturation); 94°C
for 30 s (amplification), 55°C for 45 s, 72°C for 90 s; and 72°C for 10 min (extension).
3. Examine the PCR amplification results with agarose gel electrophoresis (see Note 1).
3.3. End Repair and Blunt-End Cloning
1. Add the following to PCR tubes directly to repair the PCR products (see Note 2): 1 U
of T4 DNA polymerase; 1 UL of 4 mM dNTP solution (optional; see Note 3); 5 U of T4
polynucleotide kinase (see Note 4); and 1 µL of 1 MM ATP.
2. Incubate the reaction tubes at 25°C (room temperature) for 20 min (see Notes 5 and 6).
Stop the reactions by adding 3 mL of 0.5 M EDTA, pH 8.0.
3. Incubate the reaction tubes at 70°C for 10 min to inactive the enzymes.
4. Precipitate the PCR products by adding 5 µL of 5 M NaCl and 60 µL of isopropyl alcohol
(see Notes 7, ref. 8).
5. Resuspend the DNA fragments in 20 µL of TE or water.
6. Take 2 µL of DNA (containing approx 20–50 ng of PCR product) and mix with ligase
and vector for ligation (see Note 8): 1 µL of 10× ligase buffer; 1 µL of T4 ligase;
2 µL of repaired DNA; dephosphorylated vector (60–150 ng); and add deionized H 2 O
to a final volume of 10 µL.
7. Incubate at 16°C overnight.
8. Dilute the ligation reaction fivefold in TE buffer. Use 2 µL of the diluted ligation reaction
for transformation (see Note 9).
3.4. End Repair and Sticky-End Cloning
An EcoRI site is used as an example in the following protocol Fig. 1). However,
depending on the desired cloning site, a different combination of dNTP should be
added in the “repair” reaction (see Subheading 3.4., step 2).
1. Spin through the PCR mixture (40 µL) in a pre-equilibrated Sephacryl S-400HR spin
column (see Note 10) to remove excess dNTP.
2. Add the following to the column-purified PCR fragments to generate sticky ends: 5 µL
of 10× PCR buffer; 1 U of T4 DNA polymerase; 5 U of T4 polynucleotide kinase; 1 µL
of 4 MM dCTP and dGTP; 1 µL of 1 MM ATP; and add deionized H 2 O to a final volume
of 50 µL.
3. Incubate the reaction tubes at 25°C (room temperature) for 20 min. Stop the reaction by
adding 3 µL of 0.5 M EDTA, pH 8.0.
4. Heat inactivate the enzymes by placing the reaction tubes at 70°C for 10 min.
5. Precipitate the PCR products by adding 5 µL of 5 M NaCl and 60 µL of isopropyl
alcohol (8).
6. Resuspend the DNA fragments in 20 µL of TE or water.
7. Take 2 µL of DNA (containing approx 20–50 ng of DNA) and mix with ligase and EcoRI
digested, dephosphorylated vector for cloning: 1 µL of 10× ligase buffer; 1 µL of T4
ligase; 2 µL of repaired DNA; dephosphorylated vector (60–150 ng); and add deionized
H 2 O to a final volume of 10 µL
8. Incubate at 16°C overnight.
9. Dilute the ligation reaction fivefold in TE buffer. Use 2 µL of the diluted ligation reaction
for transformation (see Note 9).
4. Notes
1. In case of multiple PCR products from a single reaction, the specific products should
be purified by gel electrophoresis based on size after repair reaction. Several different