20.109(S13):DNA extraction (Day2) - Revision history

AgiStachowiak: /* Reagent list */

2013-02-12T15:20:51Z

Reagent list

←Older revision		Revision as of 15:20, 12 February 2013
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	* QIAamp DNA Stool Mini Kit from Qiagen		* QIAamp DNA Stool Mini Kit from Qiagen
-	* Chitinase from Sigma	+	* Chitinase from Sigma (NOT part of usual Qiagen kit!)
-	**Stock solution prepared at X concentration in ~~Y solvent<font color=red>check notebook and add!</font color>~~	+	**Stock solution prepared at 0.02666 U/μL concentration in 50 mM potassium phosphate
	* Ethanol (200 proof)		* Ethanol (200 proof)

AgiStachowiak: /* Part 1: DNA extraction from bird stool, initiate */

2013-02-12T01:56:18Z

Part 1: DNA extraction from bird stool, initiate

←Older revision		Revision as of 01:56, 12 February 2013
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	#*The solution will be homogeneous but somewhat thick.		#*The solution will be homogeneous but somewhat thick.
	#Incubate 1 min longer on a tube stand (with no shaking), and then centrifuge for 5 min (20K g) or 6 min (16K g).		#Incubate 1 min longer on a tube stand (with no shaking), and then centrifuge for 5 min (20K g) or 6 min (16K g).
-	#Transfer the supernatant usually about 500 μL to a '''1.5 mL tube''' and again centrifuge 3 or 4 min as needed.	+	#Transfer the supernatant (usually about 500 μL) to a '''1.5 mL tube''' and again centrifuge 3 or 4 min as needed.
	#In a fresh '''1.5 mL tube''', dispense 15 μL proteinase K. Only then should you add 200 μL of the supernatant above followed by 200 μL of buffer AL, pipetting to mix each time. Finally, add 15 μL chitinase.		#In a fresh '''1.5 mL tube''', dispense 15 μL proteinase K. Only then should you add 200 μL of the supernatant above followed by 200 μL of buffer AL, pipetting to mix each time. Finally, add 15 μL chitinase.
	#Vortex for 15 sec (until solution is homogeneous) and incubate in the 56 °C oven for about 2 hours.		#Vortex for 15 sec (until solution is homogeneous) and incubate in the 56 °C oven for about 2 hours.

AgiStachowiak: /* For next time */

2013-02-11T02:04:21Z

For next time

←Older revision		Revision as of 02:04, 11 February 2013
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	==For next time==		==For next time==

-	#Read the paper and guiding questions for our upcoming discussion on [[20.109%28S13%29:PCR_and_paper_discussion_%28Day3%29#Part_3:_Journal_article_discussion \| (Mod 1, Day 3)]]. Spend just 10-15 minutes preparing a single slide for your part of the discussion. ~~If you are describing a figure~~, ~~you~~ should copy ~~that~~ figure into your slide~~. Whether or not you are describing a figure~~, ~~you~~ should try to come up with a concise title that ~~relates~~ the main conclusion to be drawn from that slide.	+	#Read the [http://www.ncbi.nlm.nih.gov/pubmed/20668239 paper] and guiding questions for our upcoming discussion on [[20.109%28S13%29:PCR_and_paper_discussion_%28Day3%29#Part_3:_Journal_article_discussion \| (Mod 1, Day 3)]]. Spend just 10-15 minutes preparing a single slide for your part of the discussion. For now, aim for two things: (1) You should copy the figure you will be presenting into your slide, and (2) You should try to come up with a concise title that states the main conclusion to be drawn from that slide.

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

AgiStachowiak: /* Reagent list */

2013-02-10T14:59:47Z

Reagent list

←Older revision		Revision as of 14:59, 10 February 2013
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	* QIAamp DNA Stool Mini Kit from Qiagen		* QIAamp DNA Stool Mini Kit from Qiagen
	* Chitinase from Sigma		* Chitinase from Sigma
-	**Stock solution prepared at X concentration in Y solvent	+	**Stock solution prepared at X concentration in Y solvent<font color=red>check notebook and add!</font color>
	* Ethanol (200 proof)		* Ethanol (200 proof)

AgiStachowiak: /* Introduction */

2013-02-07T22:06:05Z

Introduction

←Older revision		Revision as of 22:06, 7 February 2013
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	==Introduction==		==Introduction==

-	~~Today you will begin your investigation of bacterial composition~~ in different ~~bird populations~~. ~~Specifically~~, ~~you will compare gulls~~ from ~~Cordova~~, ~~Alaska with those~~ that ~~frequent~~ a ~~local reservoir <font color=red>(where exactly~~ in ~~MA?). cont w~~/ ~~brief background about~~ why ~~this comparison~~ might be of ~~interest~~ and ~~how it fits into more cutting-edge research questions</font color>~~	+	Investigations in disease ecology help determine how pathogens transmit and cause disease, persist, and evolve in host organisms -- which may be as different as humans and birds. Pathogen, host, and environment all play roles in defining the natural history of disease. In studies of pathogens that cause zoonotic disease, which is transmitted from animals to people, we are particularly interested in defining major influences on their distribution and evolution. The Runstadler lab is currently researching the disease ecology of influenza viruses in several groups of birds and other animals.
		+
		+	Recent evidence in the burgeoning field of the microbiome suggests that the internal host environment may be a significant factor in the susceptibility/resistance of individuals, populations, or species to disease pathogens. Different species of birds, although in the same family or genera taxonomically, may utilize vastly different environments and travel through flyways that are separated by thousands of miles. The associated microbial communities and potential pathogens these species carry and encounter may therefore be different, and be a significant reason why one species is a host for a given pathogen and the other is not. We might ask: If two microbiomes are phylogenetically different, but functionally equivalent, does that mean they will be susceptible or resistant to similar pathogens? What do differences in microbiome structure mean for a bird’s ability to carry influenza virus, microsporidia, giardia species, or other gull associated microbes?
		+
		+	Increasingly, the associated microbial communities of animals are being shown to influence disease susceptibility and resistance to pathogens. As a part of Module 1, we will utilize an early approach to community profiling to gain a snapshot of the differences between two gull populations – one from Cordova, Alaska and the other from Revere, Massachusetts. Should we expect them to be equivalent hosts for viruses?

	To identify the bacteria cohabiting with individual birds, we'll preferentially extract pathogen (rather than animal) DNA from bird stool and sequence a conserved region. Specifically, we'll amplify 16S ribosomal RNA gene sequences using the universal primers we discussed last time in a polymerase chain reaction (PCR). The PCR will result in a pool of 16S sequences representing different species of bacteria present approximately in proportion to their composition in the bird stool. That is, a species that is abundantly present in stool is more likely to have its DNA amplified during the PCR than a low concentration species. The pool of 16S fragments can be cloned into a DNA vector and transformed into laboratory bacteria to produce isolated colonies. Each colony should contain identical copies of 16S DNA (i.e., representing a single species of bacteria), thus allowing us to deconvolve our pool of DNA and analyze individual sequences. Briefly, we'll perform a second DNA extraction from several colonies per original bird stool sample and find out the sequence of each DNA through a method that resembles PCR. The individual steps will make more sense as we complete each of them, and an overview of the process as a whole is shown below.		To identify the bacteria cohabiting with individual birds, we'll preferentially extract pathogen (rather than animal) DNA from bird stool and sequence a conserved region. Specifically, we'll amplify 16S ribosomal RNA gene sequences using the universal primers we discussed last time in a polymerase chain reaction (PCR). The PCR will result in a pool of 16S sequences representing different species of bacteria present approximately in proportion to their composition in the bird stool. That is, a species that is abundantly present in stool is more likely to have its DNA amplified during the PCR than a low concentration species. The pool of 16S fragments can be cloned into a DNA vector and transformed into laboratory bacteria to produce isolated colonies. Each colony should contain identical copies of 16S DNA (i.e., representing a single species of bacteria), thus allowing us to deconvolve our pool of DNA and analyze individual sequences. Briefly, we'll perform a second DNA extraction from several colonies per original bird stool sample and find out the sequence of each DNA through a method that resembles PCR. The individual steps will make more sense as we complete each of them, and an overview of the process as a whole is shown below.

AgiStachowiak: /* For next time */

2013-02-02T18:36:41Z

For next time

←Older revision		Revision as of 18:36, 2 February 2013
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	==For next time==		==For next time==
		+
		+	#Read the paper and guiding questions for our upcoming discussion on [[20.109%28S13%29:PCR_and_paper_discussion_%28Day3%29#Part_3:_Journal_article_discussion \| (Mod 1, Day 3)]]. Spend just 10-15 minutes preparing a single slide for your part of the discussion. If you are describing a figure, you should copy that figure into your slide. Whether or not you are describing a figure, you should try to come up with a concise title that relates the main conclusion to be drawn from that slide.

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

AgiStachowiak: /* Introduction */

2013-01-24T21:06:54Z

Introduction

←Older revision		Revision as of 21:06, 24 January 2013
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	DNA molecules passing through a silica (SiO2) column can be selectively retained by both chemical (e.g., charge) and physical (i.e., size) interactions with the porous, high surface area beads. When nucleic acids are diluted in a high concentration of a chaotropic salt buffer, they will tend to bind to the silica. This is because chaotropic salts (such as guanidine isothiocyanate, present in buffers AL and AW1) disrupt hydrogen-bond organization between water and macromolecules, essentially dehydrating the nucleic acids and causing them to bind to the resin. Ethanol (present in high concentrations in buffers AW1 and AW2) further precipitates the nucleic acids. The column-bound acids are washed with various buffers to remove salts and other contaminants before finally eluting in an ethanol-free, low-salt buffer in which nucleic acids are highly soluble. This final buffer is also at an increased pH to increase charge repulsion between silica and DNA that was previously screened under high salt, low pH conditions. The exact pore size and surface chemistry of the silica beads determine what sizes and kinds of nucleic acid will be bound versus washed away. After purification, two additional steps -- which we'll discuss next time -- can improve downstream performance in PCR.		DNA molecules passing through a silica (SiO2) column can be selectively retained by both chemical (e.g., charge) and physical (i.e., size) interactions with the porous, high surface area beads. When nucleic acids are diluted in a high concentration of a chaotropic salt buffer, they will tend to bind to the silica. This is because chaotropic salts (such as guanidine isothiocyanate, present in buffers AL and AW1) disrupt hydrogen-bond organization between water and macromolecules, essentially dehydrating the nucleic acids and causing them to bind to the resin. Ethanol (present in high concentrations in buffers AW1 and AW2) further precipitates the nucleic acids. The column-bound acids are washed with various buffers to remove salts and other contaminants before finally eluting in an ethanol-free, low-salt buffer in which nucleic acids are highly soluble. This final buffer is also at an increased pH to increase charge repulsion between silica and DNA that was previously screened under high salt, low pH conditions. The exact pore size and surface chemistry of the silica beads determine what sizes and kinds of nucleic acid will be bound versus washed away. After purification, two additional steps -- which we'll discuss next time -- can improve downstream performance in PCR.

-	We’ve made two minor modifications to the manufacturer's protocol today, due to the nature of our samples and our particular research question, respectively.One is using a somewhat lower volume of sample than recommended because bird stool is more concentrated than the human stool the kit was designed for. The second is ~~adding~~ chitinase in addition to proteinase K, and ~~incubating~~ at a lower temperature and for a longer time than recommended. Adding chitinase is useful for opening up pathogens with chitin walls, including the fungus microsporidia. You might be wondering why, since you will not use unknown samples for your microsporidia experiment this spring, but rather pre-purified DNA~~. The reason for this choice is that in practice we have found microsporidia isolation technically challenging, and nor can all bird samples be expected to contain microsporidia~~. However, your DNA may later be screened by the teaching faculty for analysis in this or a later semester of 20.109. We’re better off having the chance to find microsporidia than not! ~~And empirically~~, the low temperature/long time incubation increases recovery of bacterial DNA as well.	+	We’ve made two minor modifications to the manufacturer's protocol today, due to the nature of our samples and our particular research question, respectively. One is using a somewhat lower volume of sample than recommended because bird stool is more concentrated than the human stool the kit was designed for. The second is using chitinase in addition to proteinase K, and completing the enzymatic digestion at a lower temperature and for a longer time than recommended. Adding chitinase is useful for opening up pathogens with chitin walls, including the fungus microsporidia.
		+
		+	You might be wondering why we are bothering with chitinase, since you will not use unknown samples for your microsporidia experiment this spring, but rather pre-purified DNA. However, your DNA may later be screened by the teaching faculty for analysis in this or a later semester of 20.109. We’re better off having the chance to find microsporidia than not! In practice we have found microsporidia isolation technically challenging; nor can all bird samples be expected to contain microsporidia (in contrast to bacteria). Thus, we would like the best chance possible of isolating more strains of microsporidia than we now have available. Finally, empirically we have found that the low temperature/long time incubation increases recovery of bacterial DNA as well.

	==Protocols==		==Protocols==

AgiStachowiak: /* Part 1: DNA extraction from bird stool, initiate */

2013-01-21T18:12:18Z

Part 1: DNA extraction from bird stool, initiate

←Older revision		Revision as of 18:12, 21 January 2013
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	The following protocol requires many tube changes. Be sure that each tube is clearly labeled with your sample number to avoid swapping samples with your partner. There are a few other steps you can take to avoid cross-contamination: switch pipet tips at every step, keep only one tube open at a time, and avoid getting liquids on the lip of any tube or column.		The following protocol requires many tube changes. Be sure that each tube is clearly labeled with your sample number to avoid swapping samples with your partner. There are a few other steps you can take to avoid cross-contamination: switch pipet tips at every step, keep only one tube open at a time, and avoid getting liquids on the lip of any tube or column.

-	Before beginning this protocol, check the maximum spin speed of your centrifuge. Some centrifuges reach 20,000 g and others reach only 16,000 g, and the time for centrifugation will have to be adjusted proportionally. Note that ''rpm'' stands for rotations per minute while ''rcf'' stands for "relative centrifugal force." It is ''rcf'' that is equivalent to g-force, not ''rpm'', because ~~different~~ sized rotors will impart different forces at the same rotational speed.	+	Before beginning this protocol, check the maximum spin speed of your centrifuge. Some centrifuges reach 20,000 g and others reach only 16,000 g, and the time for centrifugation will have to be adjusted proportionally. Note that ''rpm'' stands for rotations per minute while ''rcf'' stands for "relative centrifugal force." It is ''rcf'' that is equivalent to g-force, not ''rpm'', because differently sized rotors will impart different forces at the same rotational speed.

	#Obtain your 100 μL bird stool sample from the teaching bench ice bucket, according to the number you are assigned on today's Talk page.		#Obtain your 100 μL bird stool sample from the teaching bench ice bucket, according to the number you are assigned on today's Talk page.
	#*Work quickly and keep the sample on your ice bucket until you have finished adding the lysis reagent.		#*Work quickly and keep the sample on your ice bucket until you have finished adding the lysis reagent.
	#Immediately add 1.4 mL of the lysis reagent (called buffer ASL) and vortex for ~ 1 min, until the solution is homogeneous.		#Immediately add 1.4 mL of the lysis reagent (called buffer ASL) and vortex for ~ 1 min, until the solution is homogeneous.
-	#*The fastest way to add the appropriate amount of ASL is to add 0.7 mL twice; that way you don't have to rotate the pipet setting in between additions.	+	#*The fastest way to add the appropriate amount of ASL is to add 0.7 mL twice with your P1000; that way you don't have to rotate the pipet setting in between additions.
	#*A few insoluble particulates may remain. Try vortexing for another 20-30 sec interval up to four more times, and stop vortexing when the sample no longer visibly changes over that interval.		#*A few insoluble particulates may remain. Try vortexing for another 20-30 sec interval up to four more times, and stop vortexing when the sample no longer visibly changes over that interval.
	#Heat at 70 °C for 5 min on the heat block at the front bench.		#Heat at 70 °C for 5 min on the heat block at the front bench.
-	#Vortex for 15 sec and centrifuge for 1 min at 20,000 rcf or 1.5 min at 16,000 rcf. Place your tubes so that weight is equally distributed in the centrifuge.	+	#Vortex for 15 sec and centrifuge for '''1 min at 20,000 rcf or 1.5 min at 16,000 rcf'''. Place your tubes so that weight is equally distributed in the centrifuge.
	#*Unfortunately, your centrifuges cannot be set for 1.25 min exactly.		#*Unfortunately, your centrifuges cannot be set for 1.25 min exactly.
	#Transfer 1.2 mL of supernatant into a fresh '''2 mL tube'''.		#Transfer 1.2 mL of supernatant into a fresh '''2 mL tube'''.

AgiStachowiak: /* Introduction */

2013-01-21T18:11:14Z

Introduction

←Older revision		Revision as of 18:11, 21 January 2013
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	Today you will begin your investigation of bacterial composition in different bird populations. Specifically, you will compare gulls from Cordova, Alaska with those that frequent a local reservoir <font color=red>(where exactly in MA?). cont w/ brief background about why this comparison might be of interest and how it fits into more cutting-edge research questions</font color>		Today you will begin your investigation of bacterial composition in different bird populations. Specifically, you will compare gulls from Cordova, Alaska with those that frequent a local reservoir <font color=red>(where exactly in MA?). cont w/ brief background about why this comparison might be of interest and how it fits into more cutting-edge research questions</font color>

-	To identify the bacteria cohabiting with individual birds, we'll preferentially extract pathogen (rather than animal) DNA from bird stool and sequence a conserved region. Specifically, we'll amplify 16S ribosomal RNA gene sequences using the universal primers we discussed last time in a polymerase chain reaction (PCR). The PCR will result in a pool of 16S sequences representing different species of bacteria present approximately in proportion to their composition in the bird stool. That is, a species that is abundantly present in stool is more likely to have its DNA amplified during the PCR than a ~~rarely present~~ species. The pool of 16S fragments can be cloned into a DNA vector and transformed into laboratory bacteria to produce isolated colonies. Each colony should contain identical copies of 16S DNA (i.e., representing a single species of bacteria), thus allowing us to deconvolve our pool of DNA and analyze individual sequences. Briefly, we'll perform a second DNA extraction from several colonies per original bird stool sample and find out the sequence of each DNA through a method that resembles PCR. The individual steps will make more sense as we complete each of them, and an overview of the process as a whole is shown below.	+	To identify the bacteria cohabiting with individual birds, we'll preferentially extract pathogen (rather than animal) DNA from bird stool and sequence a conserved region. Specifically, we'll amplify 16S ribosomal RNA gene sequences using the universal primers we discussed last time in a polymerase chain reaction (PCR). The PCR will result in a pool of 16S sequences representing different species of bacteria present approximately in proportion to their composition in the bird stool. That is, a species that is abundantly present in stool is more likely to have its DNA amplified during the PCR than a low concentration species. The pool of 16S fragments can be cloned into a DNA vector and transformed into laboratory bacteria to produce isolated colonies. Each colony should contain identical copies of 16S DNA (i.e., representing a single species of bacteria), thus allowing us to deconvolve our pool of DNA and analyze individual sequences. Briefly, we'll perform a second DNA extraction from several colonies per original bird stool sample and find out the sequence of each DNA through a method that resembles PCR. The individual steps will make more sense as we complete each of them, and an overview of the process as a whole is shown below.

	[[Image:S13-M1 experiment-overview.jpg\|thumb\|450px\|center\|'''Bacteria experiment overview.''']]		[[Image:S13-M1 experiment-overview.jpg\|thumb\|450px\|center\|'''Bacteria experiment overview.''']]

AgiStachowiak: /* Part 1: DNA extraction from bird stool, initiate */

2013-01-21T18:10:42Z

Part 1: DNA extraction from bird stool, initiate

←Older revision		Revision as of 18:10, 21 January 2013
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	#Transfer the supernatant usually about 500 μL to a '''1.5 mL tube''' and again centrifuge 3 or 4 min as needed.		#Transfer the supernatant usually about 500 μL to a '''1.5 mL tube''' and again centrifuge 3 or 4 min as needed.
	#In a fresh '''1.5 mL tube''', dispense 15 μL proteinase K. Only then should you add 200 μL of the supernatant above followed by 200 μL of buffer AL, pipetting to mix each time. Finally, add 15 μL chitinase.		#In a fresh '''1.5 mL tube''', dispense 15 μL proteinase K. Only then should you add 200 μL of the supernatant above followed by 200 μL of buffer AL, pipetting to mix each time. Finally, add 15 μL chitinase.
-	#Vortex for 15 sec (until solution is homogeneous) and incubate in the 56 °C oven for about 2 hours ~~<font color=red>[1.5??]</font color>~~.	+	#Vortex for 15 sec (until solution is homogeneous) and incubate in the 56 °C oven for about 2 hours.

	===Part 2: Writing Across the Curriculum (WAC) session===		===Part 2: Writing Across the Curriculum (WAC) session===