Streptomyces:Protocols/PCR: Difference between revisions
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{{Streptomyces}} | {{Streptomyces}} | ||
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[[Category:Protocol]] | |||
[[Category:Streptomyces]] | |||
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Total volume in a PCR tube - 50µL | Total volume in a PCR tube - 50µL | ||
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{| cellspacing="0" cellpadding="0" | {| cellspacing="0" cellpadding="0" | ||
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! style="width: | ! style="width: 200px; text-align: left;"| Promega GoTaq: || style="width: 150px;"| Ingredient || style="width: 100px;"| Stock Conc<sup>n</sup> || style="width: 100px;"| Volume || style="width: 150px;"| Final Conc<sup>n</sup> | ||
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| || colspan="4" | | | || colspan="4" | | ||
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| || dH<sub>2</sub>O || N/A || 16.75μL ||N/A | | || dH<sub>2</sub>O || N/A || 16.75μL ||N/A | ||
|- | |- | ||
| | | style="text-align: left;" | Mix and when ready <br/>to start the cycle add: | ||
|- | |- | ||
| || DNA Polymerase || 5u/μL || 0.25μL || 1.25u | | || DNA Polymerase || 5u/μL || 0.25μL || 1.25u | ||
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! style="width: | ! style="width: 200px; text-align: left;"| Roche High Fidelity: || style="width: 150px;"| Ingredient || style="width: 100px;"| Stock Conc<sup>n</sup> || style="width: 100px;"| Volume || style="width: 150px;"| Final Conc<sup>n</sup> | ||
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| || colspan="4" | | | || colspan="4" | | ||
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| || dH<sub>2</sub>O || N/A || 16.75μL || N/A | | || dH<sub>2</sub>O || N/A || 16.75μL || N/A | ||
|- | |- | ||
| | | style="text-align: left;" | Mix and when ready <br/>to start the cycle add: | ||
|- | |- | ||
| || DNA Polymerase || 5u/μL || 0.5μL || 2.5u | | || DNA Polymerase || 5u/μL || 0.5μL || 2.5u | ||
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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. | 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. | 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 | 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 T<sub>m</sub>-5°C (T<sub>m</sub>: 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. | ||
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==BioCoder version== | |||
Following is the Polymerase Chain Reaction (PCR) protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth|Vaishnavi]] | |||
====Text Output==== | |||
[[Polymerase Chain Reaction (PCR) protocol]] | |||
====Source Code==== | |||
[[Polymerase Chain Reaction (PCR) protocol - source code]] |
Latest revision as of 02:55, 19 November 2009
Protocols - PCR
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Polymerase Chain Reaction (PCR)Description
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BioCoder version
Following is the Polymerase Chain Reaction (PCR) protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi
Text Output
Polymerase Chain Reaction (PCR) protocol