Prather:Gibson CBA - Revision history

Ksolomon: /* References */

2010-08-09T00:28:20Z

References

←Older revision		Revision as of 00:28, 9 August 2010
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	<biblio>		<biblio>
	#Gibson-nmeth-2009 pmid=19363495		#Gibson-nmeth-2009 pmid=19363495
		+	//Isothermal assembly method
	#Gibson-science-2008 pmid=18218864		#Gibson-science-2008 pmid=18218864
		+	//Two step thermocycled assembly variant
	</biblio>		</biblio>
	<br />		<br />
	----		----
	[[Prather:Protocols\|Back to Prather Lab Protocols]]		[[Prather:Protocols\|Back to Prather Lab Protocols]]

Ksolomon: /* References */

2010-08-09T00:26:22Z

References

←Older revision		Revision as of 00:26, 9 August 2010
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	==References==		==References==
	<biblio>		<biblio>
-	#Gibson-nmeth-2009 pmid=19363495 ~~<br />~~	+	#Gibson-nmeth-2009 pmid=19363495
	#Gibson-science-2008 pmid=18218864		#Gibson-science-2008 pmid=18218864
	</biblio>		</biblio>

Ksolomon: /* References */

2010-08-09T00:26:08Z

References

←Older revision		Revision as of 00:26, 9 August 2010
Line 53:		Line 53:
	==References==		==References==
	<biblio>		<biblio>
-	#Gibson-nmeth-2009 pmid=19363495	+	#Gibson-nmeth-2009 pmid=19363495 <br />
-		+
	#Gibson-science-2008 pmid=18218864		#Gibson-science-2008 pmid=18218864
	</biblio>		</biblio>

Ksolomon: /* References */

2010-08-09T00:25:35Z

References

←Older revision		Revision as of 00:25, 9 August 2010
Line 54:		Line 54:
	<biblio>		<biblio>
	#Gibson-nmeth-2009 pmid=19363495		#Gibson-nmeth-2009 pmid=19363495
		+
	#Gibson-science-2008 pmid=18218864		#Gibson-science-2008 pmid=18218864
	</biblio>		</biblio>

Ksolomon: /* References */

2010-08-09T00:25:20Z

References

←Older revision		Revision as of 00:25, 9 August 2010
Line 54:		Line 54:
	<biblio>		<biblio>
	#Gibson-nmeth-2009 pmid=19363495		#Gibson-nmeth-2009 pmid=19363495
		+	#Gibson-science-2008 pmid=18218864
	</biblio>		</biblio>
	<br />		<br />
	----		----
	[[Prather:Protocols\|Back to Prather Lab Protocols]]		[[Prather:Protocols\|Back to Prather Lab Protocols]]

Ksolomon: /* Reagents */

2010-08-09T00:23:41Z

Reagents

←Older revision		Revision as of 00:23, 9 August 2010
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	:0.64 μL 10 U/μL T5 exonuclease		:0.64 μL 10 U/μL T5 exonuclease
	:20 μL 2 U/μL Phusion DNA Pol		:20 μL 2 U/μL Phusion DNA Pol
-	:0.16 μL 40 000 U/ul Taq DNA Ligase	+	:0.16 μL 40 000 U/μL Taq DNA Ligase
	:<ins>860 μL ddH2O</ins>		:<ins>860 μL ddH2O</ins>
	:1.2 ml Total		:1.2 ml Total

Ksolomon: /* Comments */

2010-08-09T00:21:13Z

Comments

←Older revision		Revision as of 00:21, 9 August 2010
Line 47:		Line 47:
	# There is a potential for mutations at the DNA boundaries which has yet to be quantified. Paper suggests 1 every 50 assemblies or so. Of the two initial assemblies I made, one had a missense mutation so sequence to verify interfaces or leave spacers (~ 50 bp or so) at the interfaces to 'absorb' these errors (KS)		# There is a potential for mutations at the DNA boundaries which has yet to be quantified. Paper suggests 1 every 50 assemblies or so. Of the two initial assemblies I made, one had a missense mutation so sequence to verify interfaces or leave spacers (~ 50 bp or so) at the interfaces to 'absorb' these errors (KS)
	# I have used PCRs as is (with PCR cleanup only) and gel extracted DNA in my assemblies. PCR cleanup gives more colonies (more DNA, better quality (no agarose/QG contamination)) but also has more false positives (PCR template plasmid). False positives may be alleviated by DpnI treatment if gel extraction is not used but I haven't tested this yet (KS).		# I have used PCRs as is (with PCR cleanup only) and gel extracted DNA in my assemblies. PCR cleanup gives more colonies (more DNA, better quality (no agarose/QG contamination)) but also has more false positives (PCR template plasmid). False positives may be alleviated by DpnI treatment if gel extraction is not used but I haven't tested this yet (KS).
-	# I once inadvertently designed my primers with 20 bp homology and a 20 bp spacer from the adjacent fragment and still got accurate plasmids. Possible to use ~~shorter primers~~ if desired (KS)	+	# I once inadvertently designed my primers with 20 bp homology and a 20 bp spacer from the adjacent fragment and still got accurate plasmids. Possible to use less overlap if desired (KS)

	<br />		<br />

Ksolomon: /* Comments */

2010-08-09T00:20:21Z

Comments

←Older revision		Revision as of 00:20, 9 August 2010
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	# When preparing the isothermal reaction mix, add the PEG slowly to liquid. If added too quickly it will form a plug which will make mixing difficult (KS)		# When preparing the isothermal reaction mix, add the PEG slowly to liquid. If added too quickly it will form a plug which will make mixing difficult (KS)
-	# The initial paper suggests that 10 - 100 ng of total DNA be used for assemblies. I've gone as high as 170 ng without any ill effects.	+	# The initial paper suggests that 10 - 100 ng of total DNA be used for assemblies. I've gone as high as 170 ng without any ill effects. (KS)
	# Have successfully used for a two way and three way ligation (KS)		# Have successfully used for a two way and three way ligation (KS)
	# There is a potential for mutations at the DNA boundaries which has yet to be quantified. Paper suggests 1 every 50 assemblies or so. Of the two initial assemblies I made, one had a missense mutation so sequence to verify interfaces or leave spacers (~ 50 bp or so) at the interfaces to 'absorb' these errors (KS)		# There is a potential for mutations at the DNA boundaries which has yet to be quantified. Paper suggests 1 every 50 assemblies or so. Of the two initial assemblies I made, one had a missense mutation so sequence to verify interfaces or leave spacers (~ 50 bp or so) at the interfaces to 'absorb' these errors (KS)

Ksolomon: /* References */

2010-08-09T00:19:56Z

References

←Older revision		Revision as of 00:19, 9 August 2010
Line 55:		Line 55:
	#Gibson-nmeth-2009 pmid=19363495		#Gibson-nmeth-2009 pmid=19363495
	</biblio>		</biblio>
		+	<br />
	----		----
	[[Prather:Protocols\|Back to Prather Lab Protocols]]		[[Prather:Protocols\|Back to Prather Lab Protocols]]

Ksolomon: /* Comments */

2010-08-09T00:19:34Z

Comments

←Older revision		Revision as of 00:19, 9 August 2010
Line 45:		Line 45:
	# The initial paper suggests that 10 - 100 ng of total DNA be used for assemblies. I've gone as high as 170 ng without any ill effects.		# The initial paper suggests that 10 - 100 ng of total DNA be used for assemblies. I've gone as high as 170 ng without any ill effects.
	# Have successfully used for a two way and three way ligation (KS)		# Have successfully used for a two way and three way ligation (KS)
-	#There is a potential for mutations at the DNA boundaries which has yet to be quantified. Paper suggests 1 every 50 assemblies or so. Of the two initial assemblies I made, one had a missense mutation so sequence to verify interfaces or leave spacers (~ 50 bp or so) at the interfaces to 'absorb' these errors (KS)	+	# There is a potential for mutations at the DNA boundaries which has yet to be quantified. Paper suggests 1 every 50 assemblies or so. Of the two initial assemblies I made, one had a missense mutation so sequence to verify interfaces or leave spacers (~ 50 bp or so) at the interfaces to 'absorb' these errors (KS)
-	# I have used PCRs as is (with PCR cleanup only) and gel extracted DNA in my assemblies. PCR cleanup gives more colonies (more DNA, better quality (no agarose/QG contamination)) but also has more false positives (PCR template plasmid). False ~~positive could~~ be alleviated by DpnI treatment if gel extraction is not used but I haven't tested this yet (KS).	+	# I have used PCRs as is (with PCR cleanup only) and gel extracted DNA in my assemblies. PCR cleanup gives more colonies (more DNA, better quality (no agarose/QG contamination)) but also has more false positives (PCR template plasmid). False positives may be alleviated by DpnI treatment if gel extraction is not used but I haven't tested this yet (KS).
-	# I inadvertently designed my primers with 20 bp homology and a 20 bp spacer from the adjacent fragment and still got accurate plasmids. Possible to use shorter primers if desired (KS)	+	# I once inadvertently designed my primers with 20 bp homology and a 20 bp spacer from the adjacent fragment and still got accurate plasmids. Possible to use shorter primers if desired (KS)

	<br />		<br />