20.109(S13):Bacterial amplification of DNA (Day3) - Revision history

AgiStachowiak: /* For next time */

2013-03-19T19:29:34Z

For next time

←Older revision		Revision as of 19:29, 19 March 2013
Line 136:		Line 136:
	'''Please clearly show all your work and reasoning throughout this assignment. Parts A through C should be completed for ''two different'' digests: X#Z and the reference mutant'''.		'''Please clearly show all your work and reasoning throughout this assignment. Parts A through C should be completed for ''two different'' digests: X#Z and the reference mutant'''.

-	(A) First, use the [https://www.neb.com/tools-and-resources/interactive-tools/enzyme-finder NEB site] to determine the appropriate buffer and temperature for your reaction.	+	(A) First, use the [https://www.neb.com/tools-and-resources/interactive-tools/enzyme-finder NEB site] to determine the appropriate buffer and temperature for your reaction. Searching for your enzyme by name is probably easiest.

	(B) Next, you should explicitly show how the digest distinguishes between parent IPC and mutant IPC. In other words, what are the expected band sizes for each plasmid upon digestion?		(B) Next, you should explicitly show how the digest distinguishes between parent IPC and mutant IPC. In other words, what are the expected band sizes for each plasmid upon digestion?

AgiStachowiak: /* For next time */

2013-03-19T19:29:06Z

For next time

←Older revision		Revision as of 19:29, 19 March 2013
Line 136:		Line 136:
	'''Please clearly show all your work and reasoning throughout this assignment. Parts A through C should be completed for ''two different'' digests: X#Z and the reference mutant'''.		'''Please clearly show all your work and reasoning throughout this assignment. Parts A through C should be completed for ''two different'' digests: X#Z and the reference mutant'''.

-	(A) First, use the [~~http~~://www.neb.com/~~nebecomm~~/~~tech_reference~~/~~restriction_enzymes/buffer_activity_restriction_enzymes.asp?~~ NEB site] to determine the appropriate buffer and temperature for your reaction.	+	(A) First, use the [https://www.neb.com/tools-and-resources/interactive-tools/enzyme-finder NEB site] to determine the appropriate buffer and temperature for your reaction.

	(B) Next, you should explicitly show how the digest distinguishes between parent IPC and mutant IPC. In other words, what are the expected band sizes for each plasmid upon digestion?		(B) Next, you should explicitly show how the digest distinguishes between parent IPC and mutant IPC. In other words, what are the expected band sizes for each plasmid upon digestion?

AgiStachowiak: /* For next time */

2013-03-18T23:01:44Z

For next time

←Older revision		Revision as of 23:01, 18 March 2013
Line 121:		Line 121:

	The following assignment will prepare you to set up your digestion reactions next time. On the whole, Day 4 has a tendency to run long, so reading ahead and completing as many calculations as possible in advance will be of help in making the day run smoothly.		The following assignment will prepare you to set up your digestion reactions next time. On the whole, Day 4 has a tendency to run long, so reading ahead and completing as many calculations as possible in advance will be of help in making the day run smoothly.
		+
		+	[[Image:20109_pRSET-IPC-ParentMap.jpg\|thumb\|250px\|left\|IPC plasmid map (Q2)]]
		+	[[Image:20109_pRSET-IPC-MutantMap.jpg\|thumb\|250px\|center\|Mutant plasmid map (Q2)]]
		+	<br style="clear:both;"/>

	The pRSET plasmid with inverse pericam insert, or pRSET-IPC, is 4141 basepairs long and its GenBank file is linked [[Media: S12-M2-20109_pRSET-IPC.gb\| here]]. According to the [[Media:S12_pRSET-IPC_1or2-cutters_NEBonly.pdf \|cutters list]] that you used on Day 1, restriction site ''PvuI'' occurs at ~1655 bp, and again at ~2700 bp into pRSET-IPC. Thus, digesting this parental plasmid with the ''PvuI'' enzyme should result in two linear fragments of DNA, with about 1045 and 3095 bp sizes.		The pRSET plasmid with inverse pericam insert, or pRSET-IPC, is 4141 basepairs long and its GenBank file is linked [[Media: S12-M2-20109_pRSET-IPC.gb\| here]]. According to the [[Media:S12_pRSET-IPC_1or2-cutters_NEBonly.pdf \|cutters list]] that you used on Day 1, restriction site ''PvuI'' occurs at ~1655 bp, and again at ~2700 bp into pRSET-IPC. Thus, digesting this parental plasmid with the ''PvuI'' enzyme should result in two linear fragments of DNA, with about 1045 and 3095 bp sizes.

AgiStachowiak: /* For next time */

2013-03-18T23:00:00Z

For next time

←Older revision		Revision as of 23:00, 18 March 2013
Line 130:		Line 130:
	Use the tabulated information on the [[Talk:20.109%28S13%29:Design_mutant_%28Day1%29 \| Day 1 Talk page]] along with the cutters lists from the Day 1 protocol to determine the cut sites relevant for M124S, T79P, or D24H as needed. For E67K, the students could not create an enzyme site on the 0- or 1-and-2-cutters lists. Instead, they ended up making a site for a 6-cutter (linked [[Media:20109-S12_pRSET-IPC_6-cutters.txt \| here]]). The banding pattern should nevertheless be distinguishable for the parent and mutant case, even though not all the bands will necessarily be visible.		Use the tabulated information on the [[Talk:20.109%28S13%29:Design_mutant_%28Day1%29 \| Day 1 Talk page]] along with the cutters lists from the Day 1 protocol to determine the cut sites relevant for M124S, T79P, or D24H as needed. For E67K, the students could not create an enzyme site on the 0- or 1-and-2-cutters lists. Instead, they ended up making a site for a 6-cutter (linked [[Media:20109-S12_pRSET-IPC_6-cutters.txt \| here]]). The banding pattern should nevertheless be distinguishable for the parent and mutant case, even though not all the bands will necessarily be visible.

-	'''Please clearly show all your work and reasoning throughout this assignment. Parts A through C should be completed for ''two different'' digests: X#Z and the reference mutant'''	+	'''Please clearly show all your work and reasoning throughout this assignment. Parts A through C should be completed for ''two different'' digests: X#Z and the reference mutant'''.

	(A) First, use the [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp? NEB site] to determine the appropriate buffer and temperature for your reaction.		(A) First, use the [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp? NEB site] to determine the appropriate buffer and temperature for your reaction.

AgiStachowiak: /* For next time */

2013-03-18T22:59:29Z

For next time

←Older revision		Revision as of 22:59, 18 March 2013
Line 128:		Line 128:
	For this assignment, you should plan restriction enzyme digests that allow you to distinguish parental and mutant pRSET-IPC for your X#Z mutation and for your reference mutant. You are probably best off doing a single enzyme digest for each mutant in this particular experiment. For example, above you would digest with ''PvuI''. However, in other kinds of experiments (notably cloning) using two enzymes per digest can give more information. We will discuss these types of digests in a future pre-lab if there is time.		For this assignment, you should plan restriction enzyme digests that allow you to distinguish parental and mutant pRSET-IPC for your X#Z mutation and for your reference mutant. You are probably best off doing a single enzyme digest for each mutant in this particular experiment. For example, above you would digest with ''PvuI''. However, in other kinds of experiments (notably cloning) using two enzymes per digest can give more information. We will discuss these types of digests in a future pre-lab if there is time.

-	Use the tabulated information on the [[Talk:20.109%28S13%29:~~Start-up_protein_engineering_~~%28Day1%29 \| Day 1 Talk page]] along with the cutters lists from the Day 1 protocol to determine the cut sites relevant for M124S, T79P, or D24H as needed. For E67K, the students could not create an enzyme site on the 0- or 1-and-2-cutters lists. Instead, they ended up making a site for a 6-cutter (linked [[Media:20109-S12_pRSET-IPC_6-cutters.txt \| here]]). The banding pattern should nevertheless be distinguishable for the parent and mutant case, even though not all the bands will necessarily be visible.	+	Use the tabulated information on the [[Talk:20.109%28S13%29:Design_mutant_%28Day1%29 \| Day 1 Talk page]] along with the cutters lists from the Day 1 protocol to determine the cut sites relevant for M124S, T79P, or D24H as needed. For E67K, the students could not create an enzyme site on the 0- or 1-and-2-cutters lists. Instead, they ended up making a site for a 6-cutter (linked [[Media:20109-S12_pRSET-IPC_6-cutters.txt \| here]]). The banding pattern should nevertheless be distinguishable for the parent and mutant case, even though not all the bands will necessarily be visible.

	'''Please clearly show all your work and reasoning throughout this assignment. Parts A through C should be completed for ''two different'' digests: X#Z and the reference mutant'''		'''Please clearly show all your work and reasoning throughout this assignment. Parts A through C should be completed for ''two different'' digests: X#Z and the reference mutant'''

AgiStachowiak: /* For next time */

2013-03-18T22:58:46Z

For next time

←Older revision		Revision as of 22:58, 18 March 2013
Line 119:		Line 119:

	==For next time==		==For next time==
-
-	~~<font color=red>need to revise, with apologies</font color>~~
-
-	~~Digestion plan needs to be ready for D4, not D5! Focus on that. Try to revise slightly for length.~~

	The following assignment will prepare you to set up your digestion reactions next time. On the whole, Day 4 has a tendency to run long, so reading ahead and completing as many calculations as possible in advance will be of help in making the day run smoothly.		The following assignment will prepare you to set up your digestion reactions next time. On the whole, Day 4 has a tendency to run long, so reading ahead and completing as many calculations as possible in advance will be of help in making the day run smoothly.

-	1. The pRSET plasmid with inverse pericam insert, or pRSET-IPC, is 4141 basepairs long and its GenBank file is linked [[Media: S12-M2-20109_pRSET-IPC.gb\| here]]. According to the [[Media:S12_pRSET-IPC_1or2-cutters_NEBonly.pdf \|cutters list]] that you used on Day 1, restriction site ''PvuI'' occurs at ~1655 bp, and again at ~2700 bp into pRSET-IPC. Thus, digesting this parental plasmid with the ''PvuI'' enzyme should result in two linear fragments of DNA, with about 1045 and 3095 bp sizes.	+	The pRSET plasmid with inverse pericam insert, or pRSET-IPC, is 4141 basepairs long and its GenBank file is linked [[Media: S12-M2-20109_pRSET-IPC.gb\| here]]. According to the [[Media:S12_pRSET-IPC_1or2-cutters_NEBonly.pdf \|cutters list]] that you used on Day 1, restriction site ''PvuI'' occurs at ~1655 bp, and again at ~2700 bp into pRSET-IPC. Thus, digesting this parental plasmid with the ''PvuI'' enzyme should result in two linear fragments of DNA, with about 1045 and 3095 bp sizes.

	A silent mutation can be introduced that results in a new ''PvuI'' site at the 341<sup>st</sup>-342<sup>nd</sup> residues of inverse pericam (ATT → ATC and TAC → GAC) , or approximately the 1020<sup>th</sup> basepair of IPC. When IPC is inserted into pRSET, its starting point is ~200bp into the pRSET plasmid. Thus, if the mutated pRSET-IPC plasmid is digested with ''PvuI'', three linear fragments of DNA are the result: 440, 1045, and 2655 bp. To understand these calculations, see also the plasmid maps above. '''Make sure you can reproduce the numbers in this example before proceeding with your own samples.''' Plasmid maps can be made using the ''Enzyme Selector'' and ''Graphic Map'' functions in ApE.		A silent mutation can be introduced that results in a new ''PvuI'' site at the 341<sup>st</sup>-342<sup>nd</sup> residues of inverse pericam (ATT → ATC and TAC → GAC) , or approximately the 1020<sup>th</sup> basepair of IPC. When IPC is inserted into pRSET, its starting point is ~200bp into the pRSET plasmid. Thus, if the mutated pRSET-IPC plasmid is digested with ''PvuI'', three linear fragments of DNA are the result: 440, 1045, and 2655 bp. To understand these calculations, see also the plasmid maps above. '''Make sure you can reproduce the numbers in this example before proceeding with your own samples.''' Plasmid maps can be made using the ''Enzyme Selector'' and ''Graphic Map'' functions in ApE.

AgiStachowiak: /* For next time */

2013-03-18T22:58:26Z

For next time

←Older revision		Revision as of 22:58, 18 March 2013
Line 124:		Line 124:
	Digestion plan needs to be ready for D4, not D5! Focus on that. Try to revise slightly for length.		Digestion plan needs to be ready for D4, not D5! Focus on that. Try to revise slightly for length.

-	2. The ~~pRSET plasmid with inverse pericam insert, or pRSET-IPC, is 4141 basepairs long and its GenBank file is linked [[Media: S12-M2-20109_pRSET-IPC~~.~~gb\| here]]. According to~~ the ~~[[Media:S12_pRSET-IPC_1or2-cutters_NEBonly.pdf \|cutters list]] that you used on~~ Day ~~1, restriction site ''PvuI'' occurs at ~1655 bp~~, and ~~again at ~2700 bp into pRSET-IPC. Thus, digesting this parental plasmid with the ''PvuI'' enzyme should result~~ in ~~two linear fragments~~ of ~~DNA, with about 1045 and 3095 bp sizes~~.	+	The following assignment will prepare you to set up your digestion reactions next time. On the whole, Day 4 has a tendency to run long, so reading ahead and completing as many calculations as possible in advance will be of help in making the day run smoothly.

-	~~A silent mutation can be introduced that results in a new ''PvuI'' site at the 341<sup>st</sup>-342<sup>nd</sup> residues of~~ inverse pericam ~~(ATT → ATC and TAC → GAC)~~ , or ~~approximately the 1020<sup>th</sup> basepair of IPC. When~~ IPC is ~~inserted into pRSET,~~ its ~~starting point~~ is ~~~200bp~~ into ~~the~~ pRSET ~~plasmid~~. Thus, ~~if the mutated pRSET-IPC~~ plasmid ~~is digested~~ with ''PvuI''~~, three~~ linear fragments of DNA ~~are the result: 440~~, 1045, and ~~2655~~ bp. To understand these calculations, see also the plasmid maps above. Make sure you can reproduce the numbers in this example before proceeding with your own samples. Plasmid maps can be made using the ''Enzyme Selector'' and ''Graphic Map'' functions in ApE.	+	1. The pRSET plasmid with inverse pericam insert, or pRSET-IPC, is 4141 basepairs long and its GenBank file is linked [[Media: S12-M2-20109_pRSET-IPC.gb\| here]]. According to the [[Media:S12_pRSET-IPC_1or2-cutters_NEBonly.pdf \|cutters list]] that you used on Day 1, restriction site ''PvuI'' occurs at ~1655 bp, and again at ~2700 bp into pRSET-IPC. Thus, digesting this parental plasmid with the ''PvuI'' enzyme should result in two linear fragments of DNA, with about 1045 and 3095 bp sizes.

-	~~For this assignment, you should plan restriction enzyme digests~~ that ~~allow you to distinguish parental~~ and ~~mutant~~ pRSET-IPC ~~for your X#Z mutation~~ and ~~for your positive control~~. ~~You are probably best off doing a single enzyme digest for this particular experiment. However~~, ~~in other kinds of experiments (notably cloning) using two enzymes per digest~~ can ~~give more information~~. ~~We will discuss these types of digests~~ in ~~a future pre-lab if there is time~~.	+	A silent mutation can be introduced that results in a new ''PvuI'' site at the 341<sup>st</sup>-342<sup>nd</sup> residues of inverse pericam (ATT → ATC and TAC → GAC) , or approximately the 1020<sup>th</sup> basepair of IPC. When IPC is inserted into pRSET, its starting point is ~200bp into the pRSET plasmid. Thus, if the mutated pRSET-IPC plasmid is digested with ''PvuI'', three linear fragments of DNA are the result: 440, 1045, and 2655 bp. To understand these calculations, see also the plasmid maps above. '''Make sure you can reproduce the numbers in this example before proceeding with your own samples.''' Plasmid maps can be made using the ''Enzyme Selector'' and ''Graphic Map'' functions in ApE.

-	~~Use the tabulated information on the [[Talk:20.109%28S12%29:Start~~-~~up_protein_engineering_%28Day1%29 \| Day 1 Talk page]] along with the cutters lists from the Day 1 protocol to determine the cut sites relevant~~ for ~~M124S~~ and ~~T79P~~. For ~~E67K~~, ~~the students could not create an enzyme site on the 0- or 1-and-2-cutters lists~~. ~~Instead~~, ~~they ended up making a site for a 6-cutter~~ (~~linked [[Media:20109-S12_pRSET-IPC_6-cutters.txt \| here]]~~). ~~The banding pattern should nevertheless be distinguishable for the parent and mutant case, even though not all the bands~~ will ~~be visible~~.	+	For this assignment, you should plan restriction enzyme digests that allow you to distinguish parental and mutant pRSET-IPC for your X#Z mutation and for your reference mutant. You are probably best off doing a single enzyme digest for each mutant in this particular experiment. For example, above you would digest with ''PvuI''. However, in other kinds of experiments (notably cloning) using two enzymes per digest can give more information. We will discuss these types of digests in a future pre-lab if there is time.

-	~~'''Please clearly show all your work~~ and ~~reasoning throughout this assignment~~.~~'''~~	+	Use the tabulated information on the [[Talk:20.109%28S13%29:Start-up_protein_engineering_%28Day1%29 \| Day 1 Talk page]] along with the cutters lists from the Day 1 protocol to determine the cut sites relevant for M124S, T79P, or D24H as needed. For E67K, the students could not create an enzyme site on the 0- or 1-and-2-cutters lists. Instead, they ended up making a site for a 6-cutter (linked [[Media:20109-S12_pRSET-IPC_6-cutters.txt \| here]]). The banding pattern should nevertheless be distinguishable for the parent and mutant case, even though not all the bands will necessarily be visible.

-	(A~~) First, use the [http~~:~~//www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp? NEB site] to determine~~ the ~~appropriate buffer and temperature for your two reactions.~~	+	'''Please clearly show all your work and reasoning throughout this assignment. Parts A through C should be completed for ''two different'' digests: X#Z and the reference mutant'''

-	(B) ~~Next~~, ~~you should explicitly show how~~ the ~~digest distinguishes between parent and mutant~~. ~~In other words, what are~~ the ~~expected band sizes~~ for ~~each upon digestion?~~	+	(A) First, use the [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp? NEB site] to determine the appropriate buffer and temperature for your reaction.

-	(C) Finally, to prepare for Day 4 in lab, calculate the amount of enzyme and buffer needed for your digestion master mixes. Refer to [[20.109%~~28S12~~%29:Prepare_expression_system_%28Day4%29#Diagnostic_Digests \| Part 5 (first sub-part)]] of the Day 4 protocol.	+	(B) Next, you should explicitly show how the digest distinguishes between parent IPC and mutant IPC. In other words, what are the expected band sizes for each plasmid upon digestion?
		+
		+	(C) Finally, to prepare for Day 4 in lab, calculate the amount of enzyme and buffer needed for your digestion master mixes. Refer to [[20.109%28S13%29:Prepare_expression_system_%28Day4%29#Diagnostic_Digests \| Part 5 (first sub-part)]] of the Day 4 protocol.

	==Reagent list==		==Reagent list==

AgiStachowiak: /* Part 1: Agarose gel electrophoresis */

2013-03-18T22:50:03Z

Part 1: Agarose gel electrophoresis

←Older revision		Revision as of 22:50, 18 March 2013
Line 77:		Line 77:
	<br style="clear:both;"/>		<br style="clear:both;"/>

-	While the gel runs, we will have today's pre-lab lecture. During the remaining time, you can work on Parts 3 and (optional) 5, label the tubes you will need in Part 4, work on your notebooks, start the FNT assignment, etc. '''Be sure to pre-chill your 14 mL tubes on ice for at least a few min before adding competent cells to them.'''	+	While the gel runs, we will have today's pre-lab lecture. During the remaining time, you can work on Parts 3 and (optional) 5, label the tubes you will need in Part 4, work on your notebooks, start the FNT assignment (it's a little long for a weekday assignment!), etc. '''Be sure to pre-chill your 14 mL tubes on ice for at least a few min before adding competent cells to them.'''

	===Part 2: Gel analysis===		===Part 2: Gel analysis===

AgiStachowiak: /* Protocols */

2013-03-18T22:49:09Z

Protocols

←Older revision		Revision as of 22:49, 18 March 2013
Line 17:		Line 17:

	==Protocols==		==Protocols==
-
-	~~<font color=red>Several changes being made for S13; stay tuned</font color>~~

	===Part 1: Agarose gel electrophoresis===		===Part 1: Agarose gel electrophoresis===

AgiStachowiak: /* Part 5: Statistics practice (optional) */

2013-03-18T22:48:25Z

Part 5: Statistics practice (optional)

←Older revision		Revision as of 22:48, 18 March 2013
Line 113:		Line 113:
	You may find averages, standard deviations, and t-tests useful when you report on class results. (For your own unique mutant, however, you will only have one trial unless someone else has repeated that mutant.) You will also revisit these topics during Module 3.		You may find averages, standard deviations, and t-tests useful when you report on class results. (For your own unique mutant, however, you will only have one trial unless someone else has repeated that mutant.) You will also revisit these topics during Module 3.

-	You can practice ~~doing some basic statistical analysis~~ using the male and female heights that we collected during pre-lab lecture.	+	You can practice the steps below using the male and female heights that we collected during pre-lab lecture.

	#Begin by downloading the following [[Media: S09_20109_M2D5-Stats.xls \| Excel file]] as a framework to carry out the basic statistical manipulations we discussed in pre-lab lecture. The file is modified from one originally written by [http://web.mit.edu/be/people/engelward.htm Professor Bevin Engelward].		#Begin by downloading the following [[Media: S09_20109_M2D5-Stats.xls \| Excel file]] as a framework to carry out the basic statistical manipulations we discussed in pre-lab lecture. The file is modified from one originally written by [http://web.mit.edu/be/people/engelward.htm Professor Bevin Engelward].