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

470 Wang
2. Materials
2.1. PCR
1. DNA template containing the sequence of interest.
2. Oligonucleotide primers.
3. Taq DNA polymerase.
4. 10× PCR and enzymatic repair buffer: 500 mM Tris-HCl, pH 9.0, 25 mM MgCl 2 , 500 mM
NaCl, and 5 mM DTT. Commercial 10× PCR buffer also works well.
5. 1.5 mM 10× deoxynucleotides (dNTP) solution. Concentrated stock solution (100 mM ) can
be obtained from Amersham Biosciences (Piscataway, NJ) or Boehringer Mannheim
(Indianapolis, IN).
6. Gel electrophoresis and PCR equipment.
2.2. End Repair and Blunt-End Cloning
1. Enzymes (T4 DNA polymerase, T4 polynucleotide kinase, and T4 DNA ligase). Enzymes
can be purchased from Boehringer Mannheim, Invitrogen (Carlsbad, CA) or any other
provider.
2. 1 mM of ATP solution. Concentrated stock solution (100 mM) can be obtained from
Boehringer Mannheim.
3. Isopropyl alcohol.
4. Vector (blunt end and dephosphorylated).
5. 10× Ligase buffer: 660 mM Tris-HCl, pH 7.6, 66 mM MgCl 2 , 10 mM ATP, 1 mM spermidine,
and 10 mM DTT. Commercially available 10× ligase buffers also works well.
6. TE buffer: 10 mM Tris-HCl, pH 7.5, 1 mM EDTA.
7. 5 M NaCl.
2.3. End Repair and Sticky-End Cloning
1. Sephacryl S-400 spin column. A commercial spin column (MicroSpin S-400HR) can be
obtained from Pharmacia.
2. Enzymes (T4 DNA polymerase, T4 polynucleotide kinase, and T4 DNA ligase). Enzymes
can be purchased from Boehringer Mannheim, Invitrogen, or any other provider.
3. ATP and dNTPs.
4. Isopropyl alcohol.
5. Vector (digested and dephosphorylated).
6. 10× Ligase buffer: 660 mM Tris-HCl, pH 7.6, 66 mM MgCl 2 , 10 mM ATP, 1 mM spermidine,
and 10 mM DTT. Commercially available 10× ligase buffers also works well.
7. 0.5 M EDTA, pH 8.0.
8. 5 M NaCl.
3. Method
3.1. Primer Design
For blunt-end cloning, no special primer is needed. However, secondary structure
and stretch of homopolymer should be avoided. For sticky-end cloning, depending on
restriction site selected, specific sequences should be included so that compatible ends
can be generated after T4 DNA polymerase treatment (see Subheading 3.4.).
3.2. PCR
1. Prepare the following in a PCR tube: 5 µL of 10× PCR buffer; 0.25 µg of genomic DNA;
1 µM of each primer; 0.15 mM of each deoxynucleotide (5 µL of 1.5 mM stock dNTP
solution), 1 U Taq polymerase, and deionized H 2 O to a final volume of 50 µL.