Annealing primers - Revision history

Reshma P. Shetty: Annealing Primers moved to Annealing primers: case convention

2007-10-09T21:30:29Z

Annealing Primers moved to Annealing primers: case convention

←Older revision

Revision as of 21:30, 9 October 2007

Jenny T Nguyen at 18:48, 10 July 2006

2006-07-10T18:48:47Z

←Older revision		Revision as of 18:48, 10 July 2006
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	[[Category:Protocol]]		[[Category:Protocol]]
	[[Category:DNA]]		[[Category:DNA]]
		+	[[Category:In vitro]]

Jenny T Nguyen at 16:28, 10 July 2006

2006-07-10T16:28:57Z

←Older revision		Revision as of 16:28, 10 July 2006
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	[[Category:Protocol]]		[[Category:Protocol]]
-	[{Category:DNA]]	+	[[Category:DNA]]

Jenny T Nguyen at 16:28, 10 July 2006

2006-07-10T16:28:49Z

←Older revision		Revision as of 16:28, 10 July 2006
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	[[Category:Protocol]]		[[Category:Protocol]]
		+	[{Category:DNA]]

Jenny T Nguyen at 16:27, 10 July 2006

2006-07-10T16:27:04Z

←Older revision		Revision as of 16:27, 10 July 2006
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	#[[Annealing complementary primers]]<--For pieces of DNA shorter than the limit on primer length.		#[[Annealing complementary primers]]<--For pieces of DNA shorter than the limit on primer length.
	#[[Annealing and primer extension]]<--For pieces of DNA longer than the limit on primer length.		#[[Annealing and primer extension]]<--For pieces of DNA longer than the limit on primer length.
		+
		+	[[Category:Protocol]]

Reshma P. Shetty at 18:55, 26 May 2006

2006-05-26T18:55:01Z

←Older revision		Revision as of 18:55, 26 May 2006
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	Annealing primers can be used as a fast and cheap way to synthesize a short piece of DNA for which you do not have template DNA to PCR from. See [[Synthetic Biology:BioBricks/Part fabrication\|part fabrication]] for other ways to make a part (contains [[Synthetic Biology:BioBricks\|BioBrick]] specific details).		Annealing primers can be used as a fast and cheap way to synthesize a short piece of DNA for which you do not have template DNA to PCR from. See [[Synthetic Biology:BioBricks/Part fabrication\|part fabrication]] for other ways to make a part (contains [[Synthetic Biology:BioBricks\|BioBrick]] specific details).
	#[[Annealing complementary primers]]<--For pieces of DNA shorter than the limit on primer length.		#[[Annealing complementary primers]]<--For pieces of DNA shorter than the limit on primer length.
-	#[[Annealing and primer extension]]<--For pieces of DNA longer than the limit on primer ~~lenght~~	+	#[[Annealing and primer extension]]<--For pieces of DNA longer than the limit on primer length.

Barry Canton at 17:25, 11 January 2006

2006-01-11T17:25:57Z

←Older revision		Revision as of 17:25, 11 January 2006
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-	#[[Annealing complementary primers]]	+	Annealing primers can be used as a fast and cheap way to synthesize a short piece of DNA for which you do not have template DNA to PCR from. See [[Synthetic Biology:BioBricks/Part fabrication\|part fabrication]] for other ways to make a part (contains [[Synthetic Biology:BioBricks\|BioBrick]] specific details).
-	#[[Annealing and primer extension]]	+	#[[Annealing complementary primers]]<--For pieces of DNA shorter than the limit on primer length.
		+	#[[Annealing and primer extension]]<--For pieces of DNA longer than the limit on primer lenght

Reshma P. Shetty at 16:07, 1 June 2005

2005-06-01T16:07:04Z

←Older revision		Revision as of 16:07, 1 June 2005
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-	==Annealing complementary primers==	+	#[[Annealing complementary primers]]
-		+	#[[Annealing and primer extension]]
-	~~A simple and cheap way to make a short (< 100 bp) piece of DNA is to order two complementary primers from a company such as [http://www.invitrogen.com Invitrogen~~].	+
-		+
-	*When the primers arrive, redissolve them in 50 mM Tris buffer to yield a concentration of ~800 ng/μl.	+
-		+
-	*For the annealing mix one recipe that works is as follows -	+
-	**4 μL of each of the concentrated primers.	+
-	**4 μL of salt solution (10 mM NaCl)	+
-	**28 μL of water	+
-		+
-	*The salt shields the negative charges on the single-stranded DNA molecules, allowing them to come close enough to bind.	+
-		+
-	*Anneal the primers by heating them at least 5°C above their melting point and cooling them down slowly in stages using a [[Thermocycler]~~]. Melting temperature calculations can best be done using software such as [[VectorNTI]] or data may come with the primers themselves.~~	+
-		+
-	*A simpler approach is to add the above mix in a PCR tube to a beaker of boiling water and just allow the water to cool down naturally. Most primers pairs with length less than 100bp should be fully melted at 100<sup>o</sup>C and certainly any non-specific binding should be melted at that temperature.	+
-		+
-	*Unless you have ordered your primers with 5' phosphate added you will probably improve the efficiency of any subsequent cloning steps by using adding the 5' phosphate using a protocol such as [[~~PNK Treatment of DNA Ends]]~~	+
-		+
-	==Annealing and primer extension==	+
-		+
-	~~This protocol uses annealing and primer extension to generate a short fragment of DNA (~100 bp). The DNA fragment is prepared for cloning by restriction digest.~~	+
-		+
-	~~===Materials===~~	+
-		+
-	*Two oligos which overlap by ~20 bp and have restriction enzyme sites at the 5' ends as in the diagram below. See [[Restriction Digest]~~] for notes on cutting near the ends of linear DNA fragments.~~	+
-		+
-	~~Oligo 1:    5' ---RE site-------------------------------- 3'<br>~~	+
-	Oligo 2:                                        3' --------------------------------RE site--- 5'	+
-		+
-	*[http://www.neb.com/nebecomm/products/productM0212.asp Klenow 3'<math>\rightarrow</math>5' exo<sup>-</sup> polymerase]	+
-	*dNTPs (25 mM each dNTP in stock)	+
-	*Restriction enzyme(s)	+
-	*Restriction enzyme buffer	+
-	*BSA	+
-		+
-	~~===Calculating amount of oligo for reaction===~~	+
-		+
-	~~''This should be checked for errors'' -[[User:Rshetty\|Reshma]] 19:03, 12 May 2005 (EDT)~~	+
-		+
-	~~<math> \rm{X\ L\ oligo} = \frac{\frac{Y\ g\ oligo}{(330\ g/mol\ of\ nt)(W\ nt/oligo)}\ mol\ of\ oligo}{Z\ mol/L\ oligo\ stock}</math>~~	+
-		+
-	~~===Procedure===~~	+
-		+
-	~~#Dilute the two oligos to a concentration of 10 or 25 μM using H<sub>2</sub>O~~	+
-	~~#Mix the following in a 0.6 mL sterile tube~~	+
-	~~#*10 μL 10X restriction enzyme buffer~~	+
-	~~#*1 μL 100X BSA~~	+
-	~~#*X μL oligo 1 (typically 1 μg or more)~~	+
-	~~#*Y μL oligo 2 (typically 1 μg or more)~~	+
-	~~#*(87 - X - Y) μL deionized sterile H<sub>2</sub>O~~	+
-	#Anneal the two oligos together by either placing the mixture in a thermal cycler ([http://www.mjr.com MJ Research], PTC-200) at 94°C for 5 mins, a cool down for 0.1°C/sec to 65°C, 65°C for 5 mins, then a cool down for 0.1°C/sec to 37°C. Alternatively, the tube can be placed in a beaker of boiling water and let cool to room temperature.	+
-	~~#Add 1 μL Klenow 3'<math>\rightarrow</math>5' exo<sup>-</sup> polymerase to mixture. <br> Vortex polymerase before pipetting to ensure it is well-mixed.~~	+
-	~~#Add 1 μL dNTPS (equal to 0.25 mM final concentration of each dNTP). <br> ''Recommend using a thermal cycler for the following incubation steps.''~~	+
-	~~#Incubate 1 hr at 37°C.~~	+
-	~~#Heat inactivate polymerase by incubating at 75°C for 20 minutes. <br> ''See [[Restriction Digest]] for more information on the following steps.''~~	+
-	~~#Add 1 μL restriction enzyme(s) to mixture.~~	+
-	~~#Incubate for a minimum of 2 hrs.~~	+
-	~~#Heat inactivate restriction enzyme by incubating at 80°C for 20 mins.~~	+
-	~~#[[Purification of DNA \| Purify DNA]] as necessary~~	+
-		+
-	~~==References==~~	+
-		+
-	W. P. Stemmer, A. Crameri, K. D. Ha, T. M. Brennan, and H. L. Heyneker. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene, 164(1):49–53, 1995. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids=7590320&dopt=Abstract PubMed]	+

Reshma P. Shetty: /* Procedure */

2005-06-01T15:45:42Z

Procedure

←Older revision		Revision as of 15:45, 1 June 2005
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	#*1 μL 100X BSA		#*1 μL 100X BSA
	#*X μL oligo 1 (typically 1 μg or more)		#*X μL oligo 1 (typically 1 μg or more)
-	#*Y μL oligo 1 (typically 1 μg or more)	+	#*Y μL oligo 2 (typically 1 μg or more)
	#*(87 - X - Y) μL deionized sterile H<sub>2</sub>O		#*(87 - X - Y) μL deionized sterile H<sub>2</sub>O
	#Anneal the two oligos together by either placing the mixture in a thermal cycler ([http://www.mjr.com MJ Research], PTC-200) at 94°C for 5 mins, a cool down for 0.1°C/sec to 65°C, 65°C for 5 mins, then a cool down for 0.1°C/sec to 37°C. Alternatively, the tube can be placed in a beaker of boiling water and let cool to room temperature.		#Anneal the two oligos together by either placing the mixture in a thermal cycler ([http://www.mjr.com MJ Research], PTC-200) at 94°C for 5 mins, a cool down for 0.1°C/sec to 65°C, 65°C for 5 mins, then a cool down for 0.1°C/sec to 37°C. Alternatively, the tube can be placed in a beaker of boiling water and let cool to room temperature.

Reshma P. Shetty at 16:05, 31 May 2005

2005-05-31T16:05:30Z

←Older revision		Revision as of 16:05, 31 May 2005
Line 59:		Line 59:
	#Heat inactivate restriction enzyme by incubating at 80°C for 20 mins.		#Heat inactivate restriction enzyme by incubating at 80°C for 20 mins.
	#[[Purification of DNA \| Purify DNA]] as necessary		#[[Purification of DNA \| Purify DNA]] as necessary
		+
		+	==References==
		+
		+	W. P. Stemmer, A. Crameri, K. D. Ha, T. M. Brennan, and H. L. Heyneker. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene, 164(1):49–53, 1995. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids=7590320&dopt=Abstract PubMed]