20.109(F12): Quick Facts - Revision history

Eric Ma: /* Cloning */

2012-09-13T15:50:36Z

Cloning

←Older revision		Revision as of 15:50, 13 September 2012
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	==Cloning==		==Cloning==
-	What are different ways after purifying whole plasmid to ensure that the plasmid is exactly correct?	+	* What are different ways after purifying whole plasmid to ensure that the plasmid is exactly correct?

Eric Ma: /* Homologous Recombination */

2012-09-13T15:34:41Z

Homologous Recombination

←Older revision		Revision as of 15:34, 13 September 2012
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	==Homologous Recombination==		==Homologous Recombination==
	Basic fact check: In the "chewback" step, we leave a 3' overhang. What is the direction that the enzyme that performs the "chewback" going in?		Basic fact check: In the "chewback" step, we leave a 3' overhang. What is the direction that the enzyme that performs the "chewback" going in?
		+
		+	==Cloning==
		+	What are different ways after purifying whole plasmid to ensure that the plasmid is exactly correct?

Eric Ma at 15:12, 13 September 2012

2012-09-13T15:12:01Z

←Older revision		Revision as of 15:12, 13 September 2012
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	P.S. to all the 20.109 students, these are questions that have gone through my head, but I haven't written down an answer for. Lemme know if you've got an answer! *'''[[User:Eric Ma\|Eric Ma]] 16:47, 8 September 2012 (EDT)''':		P.S. to all the 20.109 students, these are questions that have gone through my head, but I haven't written down an answer for. Lemme know if you've got an answer! *'''[[User:Eric Ma\|Eric Ma]] 16:47, 8 September 2012 (EDT)''':
		+
		+
		+	==Homologous Recombination==
		+	Basic fact check: In the "chewback" step, we leave a 3' overhang. What is the direction that the enzyme that performs the "chewback" going in?

Eric Ma: /* Taq Polymerase */

2012-09-11T15:27:50Z

Taq Polymerase

←Older revision		Revision as of 15:27, 11 September 2012
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	** What is the probability of finding a mutation in a GFP gene PCR-ed up using Taq polymerase?		** What is the probability of finding a mutation in a GFP gene PCR-ed up using Taq polymerase?
	** If we start with 1 copy of GFP in a PCR reaction, then after 30 cycles, how many copies of GFP do we expect to have undergone one mutation (either away from WT, or reverting to WT)?		** If we start with 1 copy of GFP in a PCR reaction, then after 30 cycles, how many copies of GFP do we expect to have undergone one mutation (either away from WT, or reverting to WT)?
		+	** How many copies of GFP do we expect to have a mutation away from WT after 30 cycles, starting with 1 copy?

	* If you had a plasmid of size 3 kb, at a concentration typical of high copy plasmids after a mini prep (via Qiagen kits) (~150 ng/µL), and you added 1 µL of DNA into a PCR reaction (50 µL), given the typical conditions for a PCR, what are the concentrations of plasmid and primers respectively?		* If you had a plasmid of size 3 kb, at a concentration typical of high copy plasmids after a mini prep (via Qiagen kits) (~150 ng/µL), and you added 1 µL of DNA into a PCR reaction (50 µL), given the typical conditions for a PCR, what are the concentrations of plasmid and primers respectively?
-	* Would the concentrations be sufficient to explain why primers preferentially bind to the template compared to re-annealing of the template? If so, why? If not, why?	+	** Would the concentrations be sufficient to explain why primers preferentially bind to the template compared to re-annealing of the template? If so, why? If not, why?

	P.S. to all the 20.109 students, these are questions that have gone through my head, but I haven't written down an answer for. Lemme know if you've got an answer! *'''[[User:Eric Ma\|Eric Ma]] 16:47, 8 September 2012 (EDT)''':		P.S. to all the 20.109 students, these are questions that have gone through my head, but I haven't written down an answer for. Lemme know if you've got an answer! *'''[[User:Eric Ma\|Eric Ma]] 16:47, 8 September 2012 (EDT)''':

Eric Ma: /* Taq Polymerase */

2012-09-11T15:27:15Z

Taq Polymerase

←Older revision		Revision as of 15:27, 11 September 2012
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	** So would you use Taq for cloning purposes?		** So would you use Taq for cloning purposes?
	** What is the probability of finding a mutation in a GFP gene PCR-ed up using Taq polymerase?		** What is the probability of finding a mutation in a GFP gene PCR-ed up using Taq polymerase?
		+	** If we start with 1 copy of GFP in a PCR reaction, then after 30 cycles, how many copies of GFP do we expect to have undergone one mutation (either away from WT, or reverting to WT)?

	* If you had a plasmid of size 3 kb, at a concentration typical of high copy plasmids after a mini prep (via Qiagen kits) (~150 ng/µL), and you added 1 µL of DNA into a PCR reaction (50 µL), given the typical conditions for a PCR, what are the concentrations of plasmid and primers respectively?		* If you had a plasmid of size 3 kb, at a concentration typical of high copy plasmids after a mini prep (via Qiagen kits) (~150 ng/µL), and you added 1 µL of DNA into a PCR reaction (50 µL), given the typical conditions for a PCR, what are the concentrations of plasmid and primers respectively?

Eric Ma: /* Taq Polymerase */

2012-09-08T20:48:46Z

Taq Polymerase

←Older revision		Revision as of 20:48, 8 September 2012
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	** What is the probability of finding a mutation in a GFP gene PCR-ed up using Taq polymerase?		** What is the probability of finding a mutation in a GFP gene PCR-ed up using Taq polymerase?

-	* If you had a plasmid of size 3 kb, at a concentration typical of high copy plasmids (~150 ng/µL), and you added 1 µL of DNA into a PCR reaction (50 µL), given the typical conditions for a PCR, what are the concentrations of plasmid and primers respectively?	+	* If you had a plasmid of size 3 kb, at a concentration typical of high copy plasmids after a mini prep (via Qiagen kits) (~150 ng/µL), and you added 1 µL of DNA into a PCR reaction (50 µL), given the typical conditions for a PCR, what are the concentrations of plasmid and primers respectively?
	* Would the concentrations be sufficient to explain why primers preferentially bind to the template compared to re-annealing of the template? If so, why? If not, why?		* Would the concentrations be sufficient to explain why primers preferentially bind to the template compared to re-annealing of the template? If so, why? If not, why?

	P.S. to all the 20.109 students, these are questions that have gone through my head, but I haven't written down an answer for. Lemme know if you've got an answer! *'''[[User:Eric Ma\|Eric Ma]] 16:47, 8 September 2012 (EDT)''':		P.S. to all the 20.109 students, these are questions that have gone through my head, but I haven't written down an answer for. Lemme know if you've got an answer! *'''[[User:Eric Ma\|Eric Ma]] 16:47, 8 September 2012 (EDT)''':

Eric Ma: /* Taq Polymerase */

2012-09-08T20:48:08Z

Taq Polymerase

←Older revision		Revision as of 20:48, 8 September 2012
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	* Would the concentrations be sufficient to explain why primers preferentially bind to the template compared to re-annealing of the template? If so, why? If not, why?		* Would the concentrations be sufficient to explain why primers preferentially bind to the template compared to re-annealing of the template? If so, why? If not, why?

-	P.S. to all the 20.109 students, these are questions that have gone through my head, but I haven't written down an answer for. Lemme know if you've got an answer!*'''[[User:Eric Ma\|Eric Ma]] 16:47, 8 September 2012 (EDT)''':	+	P.S. to all the 20.109 students, these are questions that have gone through my head, but I haven't written down an answer for. Lemme know if you've got an answer! *'''[[User:Eric Ma\|Eric Ma]] 16:47, 8 September 2012 (EDT)''':

Eric Ma: /* Taq Polymerase */

2012-09-08T20:47:57Z

Taq Polymerase

←Older revision		Revision as of 20:47, 8 September 2012
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	* If you had a plasmid of size 3 kb, at a concentration typical of high copy plasmids (~150 ng/µL), and you added 1 µL of DNA into a PCR reaction (50 µL), given the typical conditions for a PCR, what are the concentrations of plasmid and primers respectively?		* If you had a plasmid of size 3 kb, at a concentration typical of high copy plasmids (~150 ng/µL), and you added 1 µL of DNA into a PCR reaction (50 µL), given the typical conditions for a PCR, what are the concentrations of plasmid and primers respectively?
	* Would the concentrations be sufficient to explain why primers preferentially bind to the template compared to re-annealing of the template? If so, why? If not, why?		* Would the concentrations be sufficient to explain why primers preferentially bind to the template compared to re-annealing of the template? If so, why? If not, why?
		+
		+	P.S. to all the 20.109 students, these are questions that have gone through my head, but I haven't written down an answer for. Lemme know if you've got an answer!*'''[[User:Eric Ma\|Eric Ma]] 16:47, 8 September 2012 (EDT)''':

Eric Ma at 20:47, 8 September 2012

2012-09-08T20:47:05Z

←Older revision		Revision as of 20:47, 8 September 2012
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	** So would you use Taq for cloning purposes?		** So would you use Taq for cloning purposes?
	** What is the probability of finding a mutation in a GFP gene PCR-ed up using Taq polymerase?		** What is the probability of finding a mutation in a GFP gene PCR-ed up using Taq polymerase?
		+
		+	* If you had a plasmid of size 3 kb, at a concentration typical of high copy plasmids (~150 ng/µL), and you added 1 µL of DNA into a PCR reaction (50 µL), given the typical conditions for a PCR, what are the concentrations of plasmid and primers respectively?
		+	* Would the concentrations be sufficient to explain why primers preferentially bind to the template compared to re-annealing of the template? If so, why? If not, why?

Eric Ma: /* Taq Polymerase */

2012-09-08T19:09:41Z

Taq Polymerase

←Older revision		Revision as of 19:09, 8 September 2012
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	* Taq has an error rate of about 1 in 9000. Reference: [http://www.ncbi.nlm.nih.gov/pubmed/2847780 an article in the journal Biochemistry, quick info can be found in the abstract].		* Taq has an error rate of about 1 in 9000. Reference: [http://www.ncbi.nlm.nih.gov/pubmed/2847780 an article in the journal Biochemistry, quick info can be found in the abstract].
	** So would you use Taq for cloning purposes?		** So would you use Taq for cloning purposes?
-	** What is the probability of finding a mutation in a GFP gene?	+	** What is the probability of finding a mutation in a GFP gene PCR-ed up using Taq polymerase?