| Oligonucleotide Primers (100 micromolar) |
| Vent DNA Polymerase |
| 10X Thermopol Buffer, 1X = | 10 mM KCL 20 mM Tris-HCl (pH 8.8 at 25°C) 10 mM (NH4)2SO4 2 mM MgSO4 0.1% Triton X-100 |
| 100 mM MgSO4 |
| Nucleotides (1.25 mM dNTP mix) |
| Mineral Oil |
| Luria Broth = | 10 g Bactotryptone 5 g Yeast Extract 10 g NaCl 1 Liter Final Volume |
| Sequenase DNA Polymerase |
| DNA Polymerase Termination Mixes |
| 35S-dATP |
| Qiagen QIAEX II Gel Extraction Kit |
| Qiagen Plasmid Miniprep Kit |
| Perkin Elmer Cetus DNA Thermo-Cycler |
| 1. | Usual methods are used for plasmid construction, including PCR, using an expression plasimd of choice. PCR reactions are typically 100 microliters and contain 0.1 to 100 ng of template DNA, 10 microliters 10X Thermopol buffer, 2 microliters of 100 mM MgSO4, 16 microliters of 1.25 mM nucleotide mix, 1 microliter of each oligonucleotide primer, 1 U Vent polymerase, and are adjusted to 100 microliters with sterile dH2O. The samples are overlaid with 60 microliters of mineral oil and are typically subjected to 30 cycles of denaturation (1 min, 98°C), annealing (1 min 30 s, 52°C) and extension (2 min, 72°C) using a Perkin Elmer Cetus DNA Thermo-Cycler. |
| 2. | Overlap PCR (Ho et al. 1989) can be used to make mutants and chimeras. This method involves the amplification of two overlapping mutant fragments, followed by amplification of the overlap fragment. |
| 3. | PCR using only one mutant oligonucleotide can also be used if there is an adjacent unique restriction site. |
| 4. | PCR fragments are gel-purified and extracted from agarose gel fragments using the Qiagen QIAEX II gel extraction kit. |
| 5. | Transformations are performed using the standard CaCl2 method. |
| 6. | Single colonies are grown overnight in 1 - 2 mL of Luria Broth to make minipreps. Minipreps are prepared using the Qiagen plasmid spin prep kit or a miniprep procedure of choice. |
| 7. | Plasmids can be screened for the mutation or new junction using PCR and a oligonucleotide whose 3' end is the first base of the mutation or new junction. When the mutant oligonucleotide is used to screen candidate clones in PCR reactions together with a downstream primer, fragments will only be amplified from mutant plasmids or plasmids with the new junction. These PCR reactions require the use of a DNA polymerase that is not error-correcting, for example Taq DNA polymerase. |
| 8. | After identifying a correct plasmid, the region of the plasmid that was synthesized by PCR is sequenced to confirm the presence of the mutation or new junction and exclude the presence of any PCR-induced mutations. DNA sequence analysis is carried out using the chain termination method (Sanger et al. 1977). |
| 9. | Mutant, chimeric or truncated plasmids are then transformed into the bacterial expression host of choice using the standard CaCl2 method. |
| 1. | PCRs used to synthesize fragments can be optimized by designing the oligonucleotides used in the reaction to have a similar melting temperatures (Tm). The Tm can be calculated using the following formula (Ho et al. 1989): The annealing temperature used in the PCR reactions should be equal to the Tm. |
| 2. | The use of an error-correcting DNA polymerase, for example Vent DNA polymerase, can greatly reduced the number of PCR-induced mutations. |
| 3. | The Qiagen spin prep fillers can be reused five or more times. Wash after each use by fillings with DDW and spinning 5 - 10 sec. Repeat wash 3 - 4 times. Store dry until further use. |
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Created 30 March 1999 18:50 GMT
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Modified 29 October 2004 20:45 GMT