High GC PCR - Revision history

H Otani: Added betaine.

2019-08-30T20:13:18Z

Added betaine.

← Older revision		Revision as of 20:13, 30 August 2019
Line 4:		Line 4:
	==<strong>Tips for successful high GC PCR</strong>==		==<strong>Tips for successful high GC PCR</strong>==

	*Additives such as dimethyl sulfoxide (DMSO) can be added to reaction mixtures up to a final concentration of 1-10% can help denature DNA and promote specificity.		*Additives such as dimethyl sulfoxide (DMSO) or betaine can be added to reaction mixtures up to a final concentration of 1-10% or 0.5 M, respectively, which can help denature DNA and promote specificity.

	*When designing primers, assess properties such as melting temperature and the likelihood of secondary structure formation. A useful tool for this is Net Primer (http://www.premierbiosoft.com/netprimer/).		*When designing primers, assess properties such as melting temperature and the likelihood of secondary structure formation. A useful tool for this is Net Primer (http://www.premierbiosoft.com/netprimer/).

Sam Prudence: /* Tips for successful high GC PCR */

2019-08-09T14:45:16Z

Tips for successful high GC PCR

← Older revision		Revision as of 14:45, 9 August 2019
Line 8:		Line 8:
	*When designing primers, assess properties such as melting temperature and the likelihood of secondary structure formation. A useful tool for this is Net Primer (http://www.premierbiosoft.com/netprimer/).		*When designing primers, assess properties such as melting temperature and the likelihood of secondary structure formation. A useful tool for this is Net Primer (http://www.premierbiosoft.com/netprimer/).

	*When amplifying a region of DNA, try to find small regions of higher AT content that could form part of the primer sequence. This well help lower the Tm of the primers and reduce self-annealing and secondary structure formation.		*When amplifying a region of DNA, try to find small regions of higher AT content that could form part of the primer sequence. This well help lower the Tm of the primers and reduce self-annealing and secondary structure formation. Try to aim for a primer with no more than 75% GC content.

	*Some specific polymerases can aid high GC DNA amplification. High fidelity polymerases will help improve specificity.		*Some specific polymerases can aid high GC DNA amplification. High fidelity polymerases will help improve specificity.

	*Q5® High-Fidelity DNA Polymerase comes with a high GC enhancer that can be added to reaction mixtures up to a final concentration of 10-20% to improve specificity.		*Q5® High-Fidelity DNA Polymerase comes with a high GC enhancer that can be added to reaction mixtures up to a final concentration of 10-20% to improve specificity.


	==<strong>General protocol for ''Streptomyces'' colony PCR</strong>==		==<strong>General protocol for ''Streptomyces'' colony PCR</strong>==

Sam Prudence: /* General protocol for Streptomyces colony PCR */

2019-07-04T21:58:23Z

General protocol for Streptomyces colony PCR

← Older revision		Revision as of 21:58, 4 July 2019
Line 18:		Line 18:
	#Streak your strain of interest for single colonies on [[DNA]] medium.		#Streak your strain of interest for single colonies on [[DNA]] medium.
	#Pick a single colony and crush in 100µl 50% DMSO.		#Pick a single colony and crush in 100µl 50% DMSO.
	#Heat at 65 ~~degrees~~ for 20 minutes.		#Heat at 65ºC for ~20 minutes.
	#Add 1µl of this to 10µl of PCR mix		#Add 1µl of this to 10µl of PCR mix

Sam Prudence: /* General protocol for Streptomyces colony PCR */

2019-07-04T21:55:43Z

General protocol for Streptomyces colony PCR

← Older revision		Revision as of 21:55, 4 July 2019
Line 16:		Line 16:

	==<strong>General protocol for ''Streptomyces'' colony PCR</strong>==		==<strong>General protocol for ''Streptomyces'' colony PCR</strong>==
	#Streak your strain of interest for single colonies on [[DNA]].		#Streak your strain of interest for single colonies on [[DNA]] medium.
	#Pick a single colony and crush in 100µl 50% DMSO.		#Pick a single colony and crush in 100µl 50% DMSO.
	#Heat at 65 degrees for 20 minutes.		#Heat at 65 degrees for 20 minutes.
	#Add 1µl of this to 10µl of PCR mix		#Add 1µl of this to 10µl of PCR mix

Sam Prudence: /* General protocol for Streptomyces colony PCR */

2019-07-04T21:51:54Z

General protocol for Streptomyces colony PCR

← Older revision		Revision as of 21:51, 4 July 2019
Line 17:		Line 17:
	==<strong>General protocol for ''Streptomyces'' colony PCR</strong>==		==<strong>General protocol for ''Streptomyces'' colony PCR</strong>==
	#Streak your strain of interest for single colonies on [[DNA]].		#Streak your strain of interest for single colonies on [[DNA]].
	#Pick a single colony and crush in 100;~~micro&~~l 50% DMSO.		#Pick a single colony and crush in 100µl 50% DMSO.
	#Heat at 65 degrees for 20 minutes.		#Heat at 65 degrees for 20 minutes.
	#Add 1;~~micro&~~l of this to 10;~~micro&~~l of PCR mix		#Add 1µl of this to 10µl of PCR mix

Test at 16:04, 4 July 2019

2019-07-04T16:04:13Z

← Older revision		Revision as of 16:04, 4 July 2019
Line 15:		Line 15:


	==<strong>~~How to:~~ ''Streptomyces'' colony PCR</strong>==		==<strong>General protocol for ''Streptomyces'' colony PCR</strong>==
	#Streak your strain of interest for single colonies on [[DNA]].		#Streak your strain of interest for single colonies on [[DNA]].
	#Pick a single colony and crush in 100;micro&l 50% DMSO.		#Pick a single colony and crush in 100;micro&l 50% DMSO.
	#Heat at 65 degrees for 20 minutes.		#Heat at 65 degrees for 20 minutes.
	#Add 1;micro&l of this to 10;micro&l of PCR mix		#Add 1;micro&l of this to 10;micro&l of PCR mix

Test: /* How to:Streptomyces colony PCR */

2019-07-04T16:03:20Z

How to:Streptomyces colony PCR

← Older revision		Revision as of 16:03, 4 July 2019
Line 15:		Line 15:


	==<strong>How to:''Streptomyces'' colony PCR</strong>==		==<strong>How to: ''Streptomyces'' colony PCR</strong>==
	#Streak your strain of interest for single colonies on [[DNA]].		#Streak your strain of interest for single colonies on [[DNA]].
	#Pick a single colony and crush in 100;micro&l 50% DMSO.		#Pick a single colony and crush in 100;micro&l 50% DMSO.
	#Heat at 65 degrees for 20 minutes.		#Heat at 65 degrees for 20 minutes.
	#Add 1;micro&l of this to 10;micro&l of PCR mix		#Add 1;micro&l of this to 10;micro&l of PCR mix

Test at 16:03, 4 July 2019

2019-07-04T16:03:08Z

← Older revision		Revision as of 16:03, 4 July 2019
Line 15:		Line 15:


	==<strong>~~General protocol for~~ ''Streptomyces'' colony PCR</strong>==		==<strong>How to:''Streptomyces'' colony PCR</strong>==
	#Streak your strain of interest for single colonies on [[DNA]].		#Streak your strain of interest for single colonies on [[DNA]].
	#Pick a single colony and crush in 100;micro&l 50% DMSO.		#Pick a single colony and crush in 100;micro&l 50% DMSO.
	#Heat at 65 degrees for 20 minutes.		#Heat at 65 degrees for 20 minutes.
	#Add 1;micro&l of this to 10;micro&l of PCR mix		#Add 1;micro&l of this to 10;micro&l of PCR mix

Test at 16:01, 4 July 2019

2019-07-04T16:01:37Z

← Older revision		Revision as of 16:01, 4 July 2019
Line 1:		Line 1:
			=<strong>High GC PCR</strong>=
	Actinobacteria typically have a high GC content in their DNA. High GC DNA templates can be difficult to amplify by PCR because of stronger hydrogen bonding and possible formation of secondary structures.		Actinobacteria typically have a high GC content in their DNA. High GC DNA templates can be difficult to amplify by PCR because of stronger hydrogen bonding and possible formation of secondary structures.

	==<strong>Tips for successful high GC PCR:</strong>==		==<strong>Tips for successful high GC PCR</strong>==

	*Additives such as dimethyl sulfoxide (DMSO) can be added to reaction mixtures up to a final concentration of 1-10% can help denature DNA and promote specificity.		*Additives such as dimethyl sulfoxide (DMSO) can be added to reaction mixtures up to a final concentration of 1-10% can help denature DNA and promote specificity.

Test at 16:00, 4 July 2019

2019-07-04T16:00:51Z

← Older revision		Revision as of 16:00, 4 July 2019
Line 1:		Line 1:
	Actinobacteria typically have a high GC content in their DNA. High GC DNA templates can be difficult to amplify by PCR because of stronger hydrogen bonding and possible formation of secondary structures.		Actinobacteria typically have a high GC content in their DNA. High GC DNA templates can be difficult to amplify by PCR because of stronger hydrogen bonding and possible formation of secondary structures.

	<strong>Tips for successful high GC PCR:</strong>		==<strong>Tips for successful high GC PCR:</strong>==

	*Additives such as dimethyl sulfoxide (DMSO) can be added to reaction mixtures up to a final concentration of 1-10% can help denature DNA and promote specificity.		*Additives such as dimethyl sulfoxide (DMSO) can be added to reaction mixtures up to a final concentration of 1-10% can help denature DNA and promote specificity.
Line 14:		Line 14:


	<strong>General protocol for ''Streptomyces'' colony PCR</strong>		==<strong>General protocol for ''Streptomyces'' colony PCR</strong>==
	#Streak your strain of interest for single colonies on [[DNA]].		#Streak your strain of interest for single colonies on [[DNA]].
	#Pick a single colony and crush in 100;micro&l 50% DMSO.		#Pick a single colony and crush in 100;micro&l 50% DMSO.
	#Heat at 65 degrees for 20 minutes.		#Heat at 65 degrees for 20 minutes.
	#Add 1;micro&l of this to 10;micro&l of PCR mix		#Add 1;micro&l of this to 10;micro&l of PCR mix