Streptomyces:Protocols/PCR - Revision history

Vaishnavi Ananth: /* BioStream version */

2009-11-19T09:55:43Z

BioStream version

←Older revision		Revision as of 09:55, 19 November 2009
Line 153:		Line 153:
	\|}<br style="clear:both" />		\|}<br style="clear:both" />

-	==~~BioStream~~ version==	+	==BioCoder version==
-	Following is the Polymerase Chain Reaction (PCR) protocol in ~~BioStream~~, 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 ~~BioStream~~ (see Source code). More information about ~~BioStream~~ can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth\|Vaishnavi]]	+	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====		====Text Output====
	[[Polymerase Chain Reaction (PCR) protocol]]		[[Polymerase Chain Reaction (PCR) protocol]]
	====Source Code====		====Source Code====
	[[Polymerase Chain Reaction (PCR) protocol - source code]]		[[Polymerase Chain Reaction (PCR) protocol - source code]]

Vaishnavi Ananth at 08:44, 23 October 2009

2009-10-23T08:44:49Z

←Older revision		Revision as of 08:44, 23 October 2009
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	<!--CONTENT END-->		<!--CONTENT END-->
	\|}<br style="clear:both" />		\|}<br style="clear:both" />
		+
		+	==BioStream version==
		+	Following is the Polymerase Chain Reaction (PCR) protocol in BioStream, 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 BioStream (see Source code). More information about BioStream 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]]

Torsten Waldminghaus at 14:24, 9 December 2008

2008-12-09T14:24:15Z

←Older revision		Revision as of 14:24, 9 December 2008
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	<!--THESE CATEGORY LINES ADD THE PROTOCOL TO THE OWW PROTOCOLS PAGE-->		<!--THESE CATEGORY LINES ADD THE PROTOCOL TO THE OWW PROTOCOLS PAGE-->
	[[Category:Protocol]]		[[Category:Protocol]]
-	[[Category:~~Escherichia coli~~]]	+	[[Category:Streptomyces]]
	<!--CONTENT START-->		<!--CONTENT START-->

Streptomyces at 13:01, 14 August 2007

2007-08-14T13:01:02Z

←Older revision		Revision as of 13:01, 14 August 2007
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	<!--INSERT THE TEMPLATE HEADER-->		<!--INSERT THE TEMPLATE HEADER-->
	{{Streptomyces}}		{{Streptomyces}}
-	~~<br>~~
	{\|cellspacing="5" cellpadding="10" style="background:#000; width: 800px;"		{\|cellspacing="5" cellpadding="10" style="background:#000; width: 800px;"
	\|-valign="top"		\|-valign="top"

Streptomyces at 10:04, 10 August 2007

2007-08-10T10:04:20Z

←Older revision		Revision as of 10:04, 10 August 2007
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	\|-valign="top"		\|-valign="top"
	\|style="background:#ffffff"\|		\|style="background:#ffffff"\|
		+	<!--THESE CATEGORY LINES ADD THE PROTOCOL TO THE OWW PROTOCOLS PAGE-->
		+	[[Category:Protocol]]
		+	[[Category:Escherichia coli]]
	<!--CONTENT START-->		<!--CONTENT START-->

Streptomyces at 08:31, 10 August 2007

2007-08-10T08:31:58Z

←Older revision		Revision as of 08:31, 10 August 2007
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	Total volume in a PCR tube - 50µL		Total volume in a PCR tube - 50µL

		+	<center>
	{\| cellspacing="0" cellpadding="0"		{\| cellspacing="0" cellpadding="0"
	\|-		\|-
-	! style="width: ~~150px~~;"\| Promega GoTaq: \|\| style="width: 150px;"\| Ingredient \|\| style="width: 100px;"\| Stock Conc<sup>n</sup> \|\| style="width: 100px;"\| Volume \|\| Final Conc<sup>n</sup>	+	! 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>
	\|-		\|-
	\| \|\| 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
	\|-		\|-
-	\| ~~colspan~~="~~5" \| &nbsp~~;	+	\| style="text-align: left;" \| Mix and when ready <br/>to start the cycle add:
-	\|-	+
-	~~\| colspan="5~~" \| Mix and when ready to start the cycle add:	+
	\|-		\|-
	\| \|\| DNA Polymerase \|\| 5u/μL \|\| 0.25μL \|\| 1.25u		\| \|\| DNA Polymerase \|\| 5u/μL \|\| 0.25μL \|\| 1.25u
	\|}		\|}
		+	</center>
	<br/>		<br/>
-	<~~br/~~>	+
		+
		+	<center>
	{\| cellspacing="0" cellpadding="0"		{\| cellspacing="0" cellpadding="0"
	\|-		\|-
-	! style="width: ~~150px~~;"\| Roche High Fidelity: \|\| style="width: 150px;"\| Ingredient \|\| style="width: 100px;"\| Stock Conc<sup>n</sup> \|\| style="width: 100px;"\| Volume \|\| Final Conc<sup>n</sup>	+	! 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>
	\|-		\|-
	\| \|\| 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
	\|-		\|-
-	\| ~~colspan~~="~~5" \| &nbsp~~;	+	\| style="text-align: left;" \| Mix and when ready <br/>to start the cycle add:
-	\|-	+
-	~~\| colspan="5~~" \| Mix and when ready to start the cycle add:	+
	\|-		\|-
	\| \|\| DNA Polymerase \|\| 5u/μL \|\| 0.5μL \|\| 2.5u		\| \|\| DNA Polymerase \|\| 5u/μL \|\| 0.5μL \|\| 2.5u
	\|}		\|}
		+	</center>

Streptomyces at 12:47, 27 July 2007

2007-07-27T12:47:57Z

←Older revision		Revision as of 12:47, 27 July 2007
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	\| \|\| DNA Polymerase \|\| 5u/μL \|\| 0.25μL \|\| 1.25u		\| \|\| DNA Polymerase \|\| 5u/μL \|\| 0.25μL \|\| 1.25u
	\|}		\|}
		+	<br/>
	<br/>		<br/>
	{\| cellspacing="0" cellpadding="0"		{\| cellspacing="0" cellpadding="0"

Streptomyces at 12:45, 27 July 2007

2007-07-27T12:45:56Z

←Older revision		Revision as of 12:45, 27 July 2007
Line 142:		Line 142:
	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 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.	+	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.

Streptomyces: New page: ==Protocols - PCR== {{Streptomyces}}
{|cellspacing="5" cellpadding="10" style="background:#000; width: 800px;" |-valign="top" |style="background:#fff...

2007-07-27T12:06:03Z

New page: ==Protocols - PCR==  {{Streptomyces}} {|cellspacing="5" cellpadding="10" style="background:#000; width: 800px;" |-valign="top" |style="background:#fff...

New page

==Protocols - PCR==

{{Streptomyces}}
 
{|cellspacing="5" cellpadding="10" style="background:#000; width: 800px;"
|-valign="top"
|style="background:#ffffff"|


==Polymerase Chain Reaction (PCR)==

'''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.

{| cellspacing="0" cellpadding="0"
|-
| style="width: 150px;"| Approx. Duration: || style="width: 150px;"| || style="width: 75px;"| ||
|-
| || 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, dH2O, ice, PCR tubes, thermal cycler and program.

'''Preparation'''
 
Create a 50% DMSO solution using dH2O, 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

{| cellspacing="0" cellpadding="0"
|-
! style="width: 150px;"| Promega GoTaq: || style="width: 150px;"| Ingredient || style="width: 100px;"| Stock Concn || style="width: 100px;"| Volume || Final Concn
|-
| || colspan="4" |
----
|-
| || Buffer || 5x || 10μL || 1x
|-
| || dNTPs || 1.25mM || 10μL || 0.25mM
|-
| || MgCl2 || 25mM || 5μL || 2.5mM
|-
| || DMSO || 50% || 5μL || 5%
|-
| || Upstream Primer || 50pmol || 1μL || 1pmol
|-
| || Downstream Primer || 50pmol || 1μL || 1pmol
|-
| || Template DNA || ?? || 1μL || ~0.5μg
|-
| || dH2O || N/A || 16.75μL ||N/A
|-
| colspan="5" |  
|-
| colspan="5" | Mix and when ready to start the cycle add:
|-
| || DNA Polymerase || 5u/μL || 0.25μL || 1.25u
|}
 
{| cellspacing="0" cellpadding="0"
|-
! style="width: 150px;"| Roche High Fidelity: || style="width: 150px;"| Ingredient || style="width: 100px;"| Stock Concn || style="width: 100px;"| Volume || Final Concn
|-
| || colspan="4" |
----
|-
| || Buffer (inc MgCl2) || 10x || 5µL || 1x
|-
| || dNTPs || 1.25mM || 10µL || 0.25mM
|-
| || DMSO || 50% || 5µL || 5%
|-
| || Upstream Primer || 50pmol || 1μL || 1pmol
|-
| || Downstream Primer || 50pmol || 1μL || 1pmol
|-
| || Template DNA || ?? || 1μL || ~0.5μg
|-
| || dH2O || N/A || 16.75μL || N/A
|-
| colspan="5" |  
|-
| colspan="5" | Mix and when ready to start the cycle add:
|-
| || DNA Polymerase || 5u/μL || 0.5μL || 2.5u
|}

 
{| cellspacing="0" cellpadding="0"
|-
| An example program: || style="width: 40px;"| || style="width: 90px;"| || style="width: 100px;"| ||
|-
| || 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.


|}