Designing primers - Revision history

Tk: /* Useful primer design tools */

Tk — Thu, 15 Apr 2010 02:53:24 GMT

Useful primer design tools

←Older revision		Revision as of 02:53, 15 April 2010
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	*[http://www.ncbi.nlm.nih.gov/tools/primer-blast/ Primer-BLAST] - Web software for designing primers that combines features of both Primer3 and BLAST.		*[http://www.ncbi.nlm.nih.gov/tools/primer-blast/ Primer-BLAST] - Web software for designing primers that combines features of both Primer3 and BLAST.
	*[http://bioinformatics.org/primerx/index.htm PrimerX] - Web software for designing primers for site directed mutagenesis.		*[http://bioinformatics.org/primerx/index.htm PrimerX] - Web software for designing primers for site directed mutagenesis.
		+	*[http://flypush.imgen.bcm.tmc.edu/primer/ Primer3 multiple primer design] Web SW for designing overlapping 600 bp pcr primers for long sequences.
	*[[VectorNTI]]		*[[VectorNTI]]

Torsten Waldminghaus at 17:33, 23 February 2009

Torsten Waldminghaus — Mon, 23 Feb 2009 17:33:26 GMT

←Older revision		Revision as of 17:33, 23 February 2009
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		+	{{back to protocols}}
	==Types of Primers==		==Types of Primers==
	In general, primers are designed to identify specific locations within a long region of DNA, either plasmid or genomic. Primer binding sites are ideally unique within the range of DNA found in the reaction tube. Single primers are used to amplify and label DNA fragments for sequencing reactions, or as probes for Southern blots. Pairs of primers are used to delimit the range of DNA amplified during a PCR reaction. Because biological enzymes selectively add bases on the 3' end of primed double stranded DNA, the binding of the 3' end of the primer is especially important, while the 5' end of the primer can either bind or not, with relatively little effect on most uses of the primer. The binding of the 4-e 3' to the 5' end, the opposite from the DNA synthesis direction found in biological systems. Synthesis starts with a glass bead filled column containing a specific 3' base bound to the glass. Chemically activated nucleotides, termed phosphoramidites, are linked in 3' to 5' order to fabricate the DNA, which is then cleaved from the column using aqueous ammonia. The use of chemical synthesis provides many opportunities to build DNA fragments with unnatural bases, which can be very useful in experimental settings.		In general, primers are designed to identify specific locations within a long region of DNA, either plasmid or genomic. Primer binding sites are ideally unique within the range of DNA found in the reaction tube. Single primers are used to amplify and label DNA fragments for sequencing reactions, or as probes for Southern blots. Pairs of primers are used to delimit the range of DNA amplified during a PCR reaction. Because biological enzymes selectively add bases on the 3' end of primed double stranded DNA, the binding of the 3' end of the primer is especially important, while the 5' end of the primer can either bind or not, with relatively little effect on most uses of the primer. The binding of the 4-e 3' to the 5' end, the opposite from the DNA synthesis direction found in biological systems. Synthesis starts with a glass bead filled column containing a specific 3' base bound to the glass. Chemically activated nucleotides, termed phosphoramidites, are linked in 3' to 5' order to fabricate the DNA, which is then cleaved from the column using aqueous ammonia. The use of chemical synthesis provides many opportunities to build DNA fragments with unnatural bases, which can be very useful in experimental settings.

Rocky Cheung: /* Useful primer design tools */

Rocky Cheung — Fri, 28 Nov 2008 03:50:10 GMT

Useful primer design tools

←Older revision		Revision as of 03:50, 28 November 2008
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	*[http://perlprimer.sourceforge.net/ PerlPrimer] - Open-source, downloadable PCR primer design software <cite>Marshall</cite>		*[http://perlprimer.sourceforge.net/ PerlPrimer] - Open-source, downloadable PCR primer design software <cite>Marshall</cite>
	*[http://primer3.sourceforge.net/ Primer3] - Open-source PCR primer design software. Offers both downloadable and web versions		*[http://primer3.sourceforge.net/ Primer3] - Open-source PCR primer design software. Offers both downloadable and web versions
		+	*[http://www.ncbi.nlm.nih.gov/tools/primer-blast/ Primer-BLAST] - Web software for designing primers that combines features of both Primer3 and BLAST.
	*[http://bioinformatics.org/primerx/index.htm PrimerX] - Web software for designing primers for site directed mutagenesis.		*[http://bioinformatics.org/primerx/index.htm PrimerX] - Web software for designing primers for site directed mutagenesis.
-	*[http://www.ncbi.nlm.nih.gov/tools/primer-blast/ Primer-BLAST] - Web software for designing primers that combines feature of both Primer3 and BLAST.
	*[[VectorNTI]]		*[[VectorNTI]]

Rocky Cheung: /* Useful primer design tools */

Rocky Cheung — Fri, 28 Nov 2008 03:49:29 GMT

Useful primer design tools

←Older revision		Revision as of 03:49, 28 November 2008
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	*[http://primer3.sourceforge.net/ Primer3] - Open-source PCR primer design software. Offers both downloadable and web versions		*[http://primer3.sourceforge.net/ Primer3] - Open-source PCR primer design software. Offers both downloadable and web versions
	*[http://bioinformatics.org/primerx/index.htm PrimerX] - Web software for designing primers for site directed mutagenesis.		*[http://bioinformatics.org/primerx/index.htm PrimerX] - Web software for designing primers for site directed mutagenesis.
		+	*[http://www.ncbi.nlm.nih.gov/tools/primer-blast/ Primer-BLAST] - Web software for designing primers that combines feature of both Primer3 and BLAST.
	*[[VectorNTI]]		*[[VectorNTI]]

Rocky Cheung: /* General guidelines */

Rocky Cheung — Fri, 28 Nov 2008 03:46:52 GMT

General guidelines

←Older revision		Revision as of 03:46, 28 November 2008
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	*If you plan to cut your PCR product near the ends of the linear DNA fragment, note that some enzymes do not cut efficiently at the ends of linear DNA. So include extra bases to increase the efficiency of cutting. Many enzymes work with 4 bases supposedly but XhoI was found to require more than 4 bases (8 bases was used successfully). Thus, to be on the safe side, use 8 bases whenever possible. [http://www.neb.com/ NEB] has more information [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_linearized_vector.asp here]. Read the information at NEB carefully ... they recommend adding 4 bases to the numbers listed in their table.		*If you plan to cut your PCR product near the ends of the linear DNA fragment, note that some enzymes do not cut efficiently at the ends of linear DNA. So include extra bases to increase the efficiency of cutting. Many enzymes work with 4 bases supposedly but XhoI was found to require more than 4 bases (8 bases was used successfully). Thus, to be on the safe side, use 8 bases whenever possible. [http://www.neb.com/ NEB] has more information [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_linearized_vector.asp here]. Read the information at NEB carefully ... they recommend adding 4 bases to the numbers listed in their table.
	*[[User:tk \| Tom's]] rule of thumb is that if a PCR fails, try it again. The second time around, work a bit harder by varying the annealing temperature or something else. If it fails again, redesign your primers.		*[[User:tk \| Tom's]] rule of thumb is that if a PCR fails, try it again. The second time around, work a bit harder by varying the annealing temperature or something else. If it fails again, redesign your primers.
-	*For primers used to generate PCR products for TA cloning, see [[Knight:TOPO TA cloning#Notes\|notes]] for more information on efficient addition of 3'A to PCR products.	+	*For primers used to generate PCR products for TA cloning, see [[Knight:TOPO TA cloning#Notes\|notes]] for more information on efficient addition of 3'A to PCR products. An alternative is to use blunt end TOPO cloning if the polymerase does not add 3'A.

	==BioBrick primers==		==BioBrick primers==

Reshma P. Shetty: /* Useful primer design tools */

Reshma P. Shetty — Fri, 17 Oct 2008 06:37:47 GMT

Useful primer design tools

←Older revision		Revision as of 06:37, 17 October 2008
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	===Useful primer design tools===		===Useful primer design tools===
-	*[http://synbio.mit.edu/tools/clipboard.cgi Austin's clipboard tool] - online tool for generating the complement, reverse complement and restriction enzyme site analysis of a DNA sequence. It also translates the sequence and gives the amino acids properties.
	*[http://scitools.idtdna.com/analyzer/Applications/OligoAnalyzer/ SciTools OligoAnalyzer by IDT] - a relatively complete suite of online tools for primer analysis		*[http://scitools.idtdna.com/analyzer/Applications/OligoAnalyzer/ SciTools OligoAnalyzer by IDT] - a relatively complete suite of online tools for primer analysis
	*[http://perlprimer.sourceforge.net/ PerlPrimer] - Open-source, downloadable PCR primer design software <cite>Marshall</cite>		*[http://perlprimer.sourceforge.net/ PerlPrimer] - Open-source, downloadable PCR primer design software <cite>Marshall</cite>

Reshma P. Shetty: /* Useful primer design tools */

Reshma P. Shetty — Fri, 17 Oct 2008 06:34:18 GMT

Useful primer design tools

←Older revision		Revision as of 06:34, 17 October 2008
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	===Useful primer design tools===		===Useful primer design tools===
	*[http://synbio.mit.edu/tools/clipboard.cgi Austin's clipboard tool] - online tool for generating the complement, reverse complement and restriction enzyme site analysis of a DNA sequence. It also translates the sequence and gives the amino acids properties.		*[http://synbio.mit.edu/tools/clipboard.cgi Austin's clipboard tool] - online tool for generating the complement, reverse complement and restriction enzyme site analysis of a DNA sequence. It also translates the sequence and gives the amino acids properties.
-	*[http://scitools.idtdna.com/analyzer/Applications/OligoAnalyzer/ ~~IDT~~ SciTools OligoAnalyzer] - A relatively complete suite of online tools for primer analysis.	+	*[http://scitools.idtdna.com/analyzer/Applications/OligoAnalyzer/ SciTools OligoAnalyzer by IDT] - a relatively complete suite of online tools for primer analysis
-	*[https://catalog.invitrogen.com/index.cfm Invitrogen]	+	*[http://perlprimer.sourceforge.net/ PerlPrimer] - Open-source, downloadable PCR primer design software <cite>Marshall</cite>
-	*[http://perlprimer.sourceforge.net/ ~~Perlprimer~~] - Open-source, ~~cross-platform~~ PCR primer design software	+	*[http://primer3.sourceforge.net/ Primer3] - Open-source PCR primer design software. Offers both downloadable and web versions
-	*[http://primer3.sourceforge.net/ Primer3]	+	*[http://bioinformatics.org/primerx/index.htm PrimerX] - Web software for designing primers for site directed mutagenesis.
-	*[http://bioinformatics.org/primerx/index.htm PrimerX] - ~~This is a useful online tool~~ for designing primers for site directed mutagenesis.	+
	*[[VectorNTI]]		*[[VectorNTI]]

Reshma P. Shetty: /* Resources */

Reshma P. Shetty — Fri, 17 Oct 2008 06:33:17 GMT

Resources

←Older revision		Revision as of 06:33, 17 October 2008
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	===Oligo synthesis information===		===Oligo synthesis information===
-	*[http://www.invitrogen.com/content.cfm?pageid=10039 Invitrogen custom DNA oligo FAQ]	+
-	*[https://www.invitrogen.com/contentFrame.cfm?pageid=100&contentPage=https://invitrogen.custhelp.com/cgi-bin/invitrogen.cfg/php/enduser/std_adp.php?p_faqid=22 Invitrogen FAQ on oligo purification options]	+	Also check out Invitrogen's custom DNA oligo FAQ, FAQ on oligo purification options, and FAQ on size limit of HPLC or PAGE purified oligo
-	*[https://www.invitrogen.com/contentFrame.cfm?pageid=100&contentPage=https://invitrogen.custhelp.com/cgi-bin/invitrogen.cfg/php/enduser/std_adp.php?p_faqid=1141 Invitrogen FAQ on size limit of HPLC or PAGE purified oligo]	+

	[[Category:Protocol]]		[[Category:Protocol]]
	[[Category:DNA]]		[[Category:DNA]]
	[[Category:In vitro]]		[[Category:In vitro]]

Reshma P. Shetty: /* Resources */

Reshma P. Shetty — Fri, 17 Oct 2008 06:09:17 GMT

Resources

←Older revision		Revision as of 06:09, 17 October 2008
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	*[https://catalog.invitrogen.com/index.cfm Invitrogen]		*[https://catalog.invitrogen.com/index.cfm Invitrogen]
	*[http://perlprimer.sourceforge.net/ Perlprimer] - Open-source, cross-platform PCR primer design software		*[http://perlprimer.sourceforge.net/ Perlprimer] - Open-source, cross-platform PCR primer design software
-	*[http://~~frodo~~.wi.~~mit.edu~~/~~primer3/primer3_code.html~~ Primer3]	+	*[http://primer3.sourceforge.net/ Primer3]
	*[http://bioinformatics.org/primerx/index.htm PrimerX] - This is a useful online tool for designing primers for site directed mutagenesis.		*[http://bioinformatics.org/primerx/index.htm PrimerX] - This is a useful online tool for designing primers for site directed mutagenesis.
	*[[VectorNTI]]		*[[VectorNTI]]

Reshma P. Shetty: /* Resources */

Reshma P. Shetty — Fri, 17 Oct 2008 06:05:38 GMT

Resources

←Older revision		Revision as of 06:05, 17 October 2008
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	===Useful primer design tools===		===Useful primer design tools===
	*[http://synbio.mit.edu/tools/clipboard.cgi Austin's clipboard tool] - online tool for generating the complement, reverse complement and restriction enzyme site analysis of a DNA sequence. It also translates the sequence and gives the amino acids properties.		*[http://synbio.mit.edu/tools/clipboard.cgi Austin's clipboard tool] - online tool for generating the complement, reverse complement and restriction enzyme site analysis of a DNA sequence. It also translates the sequence and gives the amino acids properties.
-	*[http://scitools.idtdna.com/analyzer/Applications/OligoAnalyzer/] - A relatively complete suite of online tools for primer analysis.	+	*[http://scitools.idtdna.com/analyzer/Applications/OligoAnalyzer/ IDT SciTools OligoAnalyzer] - A relatively complete suite of online tools for primer analysis.
	*[https://catalog.invitrogen.com/index.cfm Invitrogen]		*[https://catalog.invitrogen.com/index.cfm Invitrogen]
-	*[http://www.bioinformatics.vg/biolinks/bioinformatics/verbose/PCR%2520and%2520Primer%2520Design.shtml List of Primer Design Software]
	*[http://perlprimer.sourceforge.net/ Perlprimer] - Open-source, cross-platform PCR primer design software		*[http://perlprimer.sourceforge.net/ Perlprimer] - Open-source, cross-platform PCR primer design software
	*[http://frodo.wi.mit.edu/primer3/primer3_code.html Primer3]		*[http://frodo.wi.mit.edu/primer3/primer3_code.html Primer3]