Streptomyces:Protocols/PCR
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Description
Polymerase Chain Reaction (PCR) is a method of amplifying a specific DNA target sequence. The cycle involves denaturing the template double stranded DNA (dsDNA) to single stranded DNA (ssDNA). Custom designed primers/oligonucleotides then anneal to their target sequence on the ssDNA. The DNA polymerase then extends the primers in a 5’ → 3’ direction, creating a complementary double strand. The cycle repeats with the denaturing of the recently synthesised dsDNA. This becomes the template to which the unused primers will anneal. A programmed thermal cycler runs through a series of specific temperatures for specific durations to achieve the amplification. Primers/oligonucleotides are designed to anneal to a specific sequence of target DNA thus determining the beginning and end of the region to be amplified, e.g. flanking a gene on genomic DNA. The DNA polymerase uses Deoxynucleotide-triphosphates (dNTPs), a mix of dATP, dCTP, dGTP & dTTP in its synthesis of the complementary strand.
| Approx. Duration: | |||
| Preparation | ~5 minutes | ||
| Setting up the PCR | ~10 minutes | ||
| Whole Protocol | ~2 hours (Depending on cycle) |
Uses
PCR is the amplification of a gene for further experimentation. For example: an amplified gene can then be digested, enabling it to be cloned into a vector. Colony PCR can then be used as a check if the ligation was successful. By using the same primers and a single colony from a transformation of a ligation; amplification of the gene would indicate that the colony had a correctly ligated construct.
Prerequisites
You should know the target sequence in order to design primers. The primers should ideally be about 20-30 nucleotides/base pairs (nt/bp) long, with little or no secondary structure formation. They should have a melting temperature (Tm ) between ~55°C and 65°C and a primer pair should have Tm >±5°C. The 3’ end of each primer needs to be designed carefully as this is where the DNA polymerase action begins.
Safety
General laboratory & molecular biology safety rules apply.
Requirements
DNA template, Two oligos/primers, DNA Taq polymerase, dNTPs Deoxynucleotides-triphosphate, Buffer, 50% DMSO(Dimethylsulphoxide)/glycerol, dH2 O, ice, PCR tubes, thermal cycler and program.
Preparation
Create a 50% DMSO solution using dH2 O, see Notes for an alternative. Ensure the primers have been dissolved in a stock solution of 500pmol then to a working concentration of 50pmol. Thaw all other solutions on ice. Label PCR tubes to be used and setup the thermal cycler.
Method
Total volume in a PCR tube - 50µL
<center> <table> <tbody> <tr> <th> Roche High Fidelity:</th> <th> Ingredient</th> <th> Stock Conc<sup>n</sup></th> <th> Volume</th> <th> Final Conc<sup>n</sup></th> </tr> <tr> <td> </td> <td> <hr /> </td> </tr> <tr> <td> </td> <td> Buffer (inc MgCl<sub>2</sub> )</td> <td> 10x</td> <td> 5µL</td> <td> 1x</td> </tr> <tr> <td> </td> <td> dNTPs</td> <td> 1.25mM</td> <td> 10µL</td> <td> 0.25mM</td> </tr> <tr> <td> </td> <td> DMSO</td> <td> 50%</td> <td> 5µL</td> <td> 5%</td> </tr> <tr> <td> </td> <td> Upstream Primer</td> <td> 50pmol</td> <td> 1μL</td> <td> 1pmol</td> </tr> <tr> <td> </td> <td> Downstream Primer</td> <td> 50pmol</td> <td> 1μL</td> <td> 1pmol</td> </tr> <tr> <td> </td> <td> Template DNA</td> <td> ??</td> <td> 1μL</td> <td> ~0.5μg</td> </tr> <tr> <td> </td> <td> dH<sub>2</sub> O</td> <td> N/A</td> <td> 16.75μL</td> <td> N/A</td> </tr> <tr> <td> Mix and when ready<br /> to start the cycle add:</td> </tr> <tr> <td> </td> <td> DNA Polymerase</td> <td> 5u/μL</td> <td> 0.5μL</td> <td> 2.5u</td> </tr> </tbody> </table> </center>
| An example program: | ||||
| 96°C | � 5 minutes | This denatures any double stranded DNA | ||
| 96°C | � 1 minute | ¯\ | First temperature of the cycle � denaturing | |
| 55°C | � 30 seconds | >25 cycles | Second temperature of the cycle - annealing | |
| 72°C | � 1 minute | _/ | Third temperature of the cycle - extending | |
| 72°C | � 5 minutes | Final extension for incomplete strands | ||
| 4°C | � hold | Finished. Hold the samples at a low temperature. |
Notes
Remove the enzyme(s) from the freezer when needed and keep all the other ingredients on ice. The buffer is required to provide a suitable chemical environment for the DNA polymerase to work in. The 50% DMSO helps to avoid secondary structures forming within a primer or the ssDNA template, sometimes a 50% glycerol solution gives better results than the standard 50% DMSO solution. Adjust the volume of the template DNA according to its concentration but remember to also adjust the volume of water added for the reaction to be in 50μL. Based on the design of the primers and the length of the target sequence a cycle can be created. The initial denaturing temperature is usually 96°C for 5 minutes followed by the actual cycle. The denaturing temperature usually stays the same 96°C or can decrease to 92°C to extend the life of the DNA polymerase; it normally last for 0.5-2 minutes. The annealing temperature is approximately Tm -5°C (Tm : melting temperature of the primers) this should avoid non-specific annealing to similar but non-identical sequences; this is for ~30 seconds. The extension temperature is always 72°C as this is the optimum temperature for DNA polymerase; the time is dependant on the length of the target, Taq DNA Polymerase has an activity ~103 base pairs per minute.
