BISC209: Lab6 - Revision history

Janet McDonough: /* LAB 6: Start the Characterization of Cultured Bacteria by 16srDNA Sequencing and Analysis & Continue Traditional ID Techniques */

2010-04-23T15:33:47Z

LAB 6: Start the Characterization of Cultured Bacteria by 16srDNA Sequencing and Analysis & Continue Traditional ID Techniques

←Older revision		Revision as of 15:33, 23 April 2010
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-	==<center>LAB 6: Start the ~~Characterization of Cultured Bacteria~~ by 16srDNA Sequencing and Analysis ~~& Continue~~ Traditional ID Techniques</center>==	+	==<center>LAB 6: Start the Genomic Id by 16srDNA Sequencing and Analysis and continue Traditional ID Techniques to Help Characterize the Cultured Bacterial Isolates </center>==
	In addition to isolating genomic DNA from a soil sample, amplifying the 16S rDNA by pcr, inserting the different DNA fragments from the pcr product into a cloning vector, transforming ''E. coli'' with your clones, and sending the transformed ''E. coli'' off to have the 16s rDNA inserts sequenced in an automatic sequencer so that you can identify a large and, we hope, representative sample of the bacterial flora in the soil community from your habitat, you also have been working, simultaneously, through traditional microbiological culturing techniques, to isolate and characterize a few of the culturable bacteria through morphology, physical, and metabolism differentiation. Look how much you have accomplished in these few short weeks!!<br><BR>		In addition to isolating genomic DNA from a soil sample, amplifying the 16S rDNA by pcr, inserting the different DNA fragments from the pcr product into a cloning vector, transforming ''E. coli'' with your clones, and sending the transformed ''E. coli'' off to have the 16s rDNA inserts sequenced in an automatic sequencer so that you can identify a large and, we hope, representative sample of the bacterial flora in the soil community from your habitat, you also have been working, simultaneously, through traditional microbiological culturing techniques, to isolate and characterize a few of the culturable bacteria through morphology, physical, and metabolism differentiation. Look how much you have accomplished in these few short weeks!!<br><BR>

Janet McDonough: /* Assignment */

2010-03-27T11:55:37Z

Assignment

←Older revision		Revision as of 11:55, 27 March 2010
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	You have already used each of these molecular tools and written about all of them as a Material and Methods section, but you haven't yet been required to explain the theory behind how each of them accomplishes each of crucial steps toward our goal of identifying unknown bacteria by genus and species name from DNA sequencing. One of the problems in using sophisticated molecular tools is that you can have a very successful lab day, yet it can be mostly "hands on, brain off". Since much of what you have been doing is pipeting, mixing, and incubating of miniscule quantities of liquid reagents that come in kits, it is easy to lose sight of what is actually happening in those tubes or spin columns at each stage. The problem of "doing without knowing" is exacerbated by kit manufacturers who make their reagents "proprietary". That prevents us from knowing exactly what's in them, making it even harder to follow the chemical or physical reactions.		You have already used each of these molecular tools and written about all of them as a Material and Methods section, but you haven't yet been required to explain the theory behind how each of them accomplishes each of crucial steps toward our goal of identifying unknown bacteria by genus and species name from DNA sequencing. One of the problems in using sophisticated molecular tools is that you can have a very successful lab day, yet it can be mostly "hands on, brain off". Since much of what you have been doing is pipeting, mixing, and incubating of miniscule quantities of liquid reagents that come in kits, it is easy to lose sight of what is actually happening in those tubes or spin columns at each stage. The problem of "doing without knowing" is exacerbated by kit manufacturers who make their reagents "proprietary". That prevents us from knowing exactly what's in them, making it even harder to follow the chemical or physical reactions.

-	Despite our use of such proprietary kits, it is possible to understand how it all works. All of these tools were discovered by scientists who published their findings. You don't, however, probably need to go to primary literature (Sanger's original paper, for example) to find out how Sanger sequencing works. There are good animations of Sanger sequencing, transformation, pcr, etc. prepared by the Dolan DNA center at [[http://www.dnalc.org/resources/animations/ \| http://www.dnalc.org/resources/animations/]]. Pay particular attention to the difference between a polymerase chain reaction and the Sanger sequencing reactions described. Note that the type of cloning described in the Dolan animations is organismal cloning---not what we are doing. We are doing molecular cloning. A good animation that describes our type of plasmid cloning is found at : [http://www.sumanasinc.com/webcontent/animations/content/plasmidcloning.html \| http://www.sumanasinc.com/webcontent/animations/content/plasmidcloning.html]. Wikipedia is also a great place to start to find out some of what you need to know for this assignment. Although it won't be difficult to find out the ~~priniples~~ behind Sanger sequencing, polymerase chain reaction, plasmid cloning, making cells chemically competent for transformation, genomic DNA isolation (which pretty much uses the ~~prinicple~~ of differential solubility of DNA in ethanol), why we picked the 16S rRNA gene for sequencing to differentiate our bacterial species, etc., it will be challenging to condense each tool to essentials in your summary. Being able to distill and write a broad outline, while understanding the specifics, will be important when you describe your experimental design in your final paper.<BR><BR>	+	Despite our use of such proprietary kits, it is possible to understand how it all works. All of these tools were discovered by scientists who published their findings. You don't, however, probably need to go to primary literature (Sanger's original paper, for example) to find out how Sanger sequencing works. There are good animations of Sanger sequencing, transformation, pcr, etc. prepared by the Dolan DNA center at [[http://www.dnalc.org/resources/animations/ \| http://www.dnalc.org/resources/animations/]]. Pay particular attention to the difference between a polymerase chain reaction and the Sanger sequencing reactions described. Note that the type of cloning described in the Dolan animations is organismal cloning---not what we are doing. We are doing molecular cloning. A good animation that describes our type of plasmid cloning is found at : [http://www.sumanasinc.com/webcontent/animations/content/plasmidcloning.html \| http://www.sumanasinc.com/webcontent/animations/content/plasmidcloning.html]. Wikipedia is also a great place to start to find out some of what you need to know for this assignment. Although it won't be difficult to find out the principles behind Sanger sequencing, polymerase chain reaction, plasmid cloning, making cells chemically competent for transformation, genomic DNA isolation (which pretty much uses the principle of differential solubility of DNA in ethanol), why we picked the 16S rRNA gene for sequencing to differentiate our bacterial species, etc., it will be challenging to condense each tool to essentials in your summary. Being able to distill and write a broad outline, while understanding the specifics, will be important when you describe your experimental design in your final paper.<BR><BR>

	The users' manuals for the Zero Blunt® TOPO® PCR Cloning Kit might be helpful in getting a better understanding of the specifics of our cloning. You can download it as a pdf file from the manufacturer, Invitrogen's web site at: [http://products.invitrogen.com/ivgn/product/K280020?CID=Search-K2800-20]<BR>		The users' manuals for the Zero Blunt® TOPO® PCR Cloning Kit might be helpful in getting a better understanding of the specifics of our cloning. You can download it as a pdf file from the manufacturer, Invitrogen's web site at: [http://products.invitrogen.com/ivgn/product/K280020?CID=Search-K2800-20]<BR>

-	'''Another good source of information is the background information found in this wiki.''' Be careful about ~~inadventent~~ plagarism.<BR><BR>	+	'''Another good source of information is the background information found in this wiki.''' Be careful about inadvertent plagarism.<BR><BR>

	Remember that this summary should be not more than a couple of pages double spaced (or 1.5spacing). If you are really good at picking out essentials and being concise, you might be able to adequately explain these molecular tools in a page. <BR><BR>		Remember that this summary should be not more than a couple of pages double spaced (or 1.5spacing). If you are really good at picking out essentials and being concise, you might be able to adequately explain these molecular tools in a page. <BR><BR>

Tucker Crum: /* Part B: PCR AMPLIFICATION of 16s rDNA from lysates prepared above */

2010-03-19T13:57:37Z

Part B: PCR AMPLIFICATION of 16s rDNA from lysates prepared above

←Older revision		Revision as of 13:57, 19 March 2010
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	\| 98C <br> 55C<br> 72C		\| 98C <br> 55C<br> 72C
	\| 10 sec <br>30 sec <br> 30 sec		\| 10 sec <br>30 sec <br> 30 sec
-	\| 20	+	\| 35
	\|-		\|-
	! Final Extension		! Final Extension

Janet McDonough: /* Part C: Culturable Bacteria Characterization by Metabolic and Physical Tests */

2010-03-16T13:07:35Z

Part C: Culturable Bacteria Characterization by Metabolic and Physical Tests

←Older revision		Revision as of 13:07, 16 March 2010
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	Link to the electronic edition of [http://0-www.springerlink.com.luna.wellesley.edu/content/?k=title%3a%28bergey%27s%29&sortorder=asc&Content+Type=Books \| Bergey's Manuals]through Springer ebooks<BR><BR>		Link to the electronic edition of [http://0-www.springerlink.com.luna.wellesley.edu/content/?k=title%3a%28bergey%27s%29&sortorder=asc&Content+Type=Books \| Bergey's Manuals]through Springer ebooks<BR><BR>

-	'''Activity 1''' Continue following the protocols for [[BISC209: Roles of soil Microbes \|Tests to determine the role of a soil isolate]]. Assess the tests carried over from LAB 5 by comparing inoculated media to uninoculated. <BR><BR>	+	'''Activity 1''' Continue following the protocols for [[BISC209: Roles of soil Microbes \|Tests to determine the role of a soil isolate]]. Assess the tests carried over from LAB 5 and 6 by comparing inoculated media to uninoculated. <BR><BR>

-	Start or continue the '''Antibiotic Production''' and/or sensitivity test ~~by inoculating on of~~ the ~~plates~~ you ~~prepared last week to~~ test ~~your~~ isolates ~~as producers. Follow~~ the ~~directions for inoculating one plate with a known set of controls and inoculate the other plate with broth cultures of your~~ isolates that ~~you want~~ to ~~test for interactions (~~sensitivity~~) with the potential antibiotic producers~~. <BR><BR>	+	Start or continue the '''Antibiotic Production''' and/or sensitivity test. If you completed the test using the control Micrococcus, Staphylococcus, and E. coli, you could test any non antibiotic producing isolates against the isolates that produced antibiotics in order to examine their sensitivity. <BR><BR>

	Check on your '''cellulolytic test plates''' (with the leaf disks). You may see a lot of "swarming" outside the area of inoculation, making it difficult to use the negative control leaf disk as a comparison or to determine which of the bacteria on the plate are responsible for changes in the leaf tissue. We will continue to watch this reaction for several more weeks but make notes about the condition of the leaf tissue and perhaps take pictures for a record. <BR><BR>		Check on your '''cellulolytic test plates''' (with the leaf disks). You may see a lot of "swarming" outside the area of inoculation, making it difficult to use the negative control leaf disk as a comparison or to determine which of the bacteria on the plate are responsible for changes in the leaf tissue. We will continue to watch this reaction for several more weeks but make notes about the condition of the leaf tissue and perhaps take pictures for a record. <BR><BR>
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	<BR><BR>		<BR><BR>

-	'''Activity 2''' Continue or add appropriate new physical, stain, enzyme, metabolic, or other tests that might help characterize your isolate, particularly those tests that provide information suggesting functional roles and/or interactions with other microbes in the ecosystem. <UL><LI>	+	'''Activity 2''' Continue or add appropriate new physical, stain, enzyme, metabolic, or other tests that might help characterize your isolate, particularly those tests that provide information suggesting functional roles and/or interactions with other microbes in the ecosystem such as motility or spore production. <UL><LI>
	'''NEW TESTS'''		'''NEW TESTS'''

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	<UL><LI>		<UL><LI>
	'''Tests for Motility''' <BR>		'''Tests for Motility''' <BR>
-	~~Every~~ isolate should be inoculated in a SIM tube. This test gives information about motility and about two other metabolic pathways. <BR>	+	By now, every isolate should be inoculated in a SIM tube. This test gives information about motility and about two other metabolic pathways. <BR>
	Find the directions in ([[BISC209: Motility \| Motility Tests]] and in [[BISC209: Enzyme tests \|Enzyme tests ]]). <BR>		Find the directions in ([[BISC209: Motility \| Motility Tests]] and in [[BISC209: Enzyme tests \|Enzyme tests ]]). <BR>
	If you want additional confirmation of motility, you can perform a wet mount technique for motility and/or the flagella stain. The directions for these tests are found in the Protocols section of the wiki at: [[BISC209: Motility]]. If you decide to perform a wet mount analysis of motility, be aware that, unfortunately, when viewing living bacterium in a wet mount at 400x magnification, it is difficult to see unstained, tiny bacteria. If you choose to try to assess motility in a wet mount, ask your instructor for help. Note: You may also use the broth culture that you set up for the cellulose degradation test and to test your isolates ability to resist antibiotics as described in [[BISC209: Roles of soil Microbes \|Tests to determine the role of a soil isolate]] protocol in this wiki can be used to make a wet mount.		If you want additional confirmation of motility, you can perform a wet mount technique for motility and/or the flagella stain. The directions for these tests are found in the Protocols section of the wiki at: [[BISC209: Motility]]. If you decide to perform a wet mount analysis of motility, be aware that, unfortunately, when viewing living bacterium in a wet mount at 400x magnification, it is difficult to see unstained, tiny bacteria. If you choose to try to assess motility in a wet mount, ask your instructor for help. Note: You may also use the broth culture that you set up for the cellulose degradation test and to test your isolates ability to resist antibiotics as described in [[BISC209: Roles of soil Microbes \|Tests to determine the role of a soil isolate]] protocol in this wiki can be used to make a wet mount.

Janet McDonough: /* Part B: PCR AMPLIFICATION of 16s rDNA from lysates prepared above */

2010-03-16T12:42:05Z

Part B: PCR AMPLIFICATION of 16s rDNA from lysates prepared above

←Older revision		Revision as of 12:42, 16 March 2010
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	{\| border="1"		{\| border="1"
	\|+		\|+
-	! Component !! amt. in a 50 μl<br>reaction !! Final Conc.	+	! Component !! amt. in a 25 μl<br>reaction !! Final Conc.
	\|-		\|-
	! Purified<BR>DNAase free <BR> Water		! Purified<BR>DNAase free <BR> Water
-	\| 13 μl	+	\| 8 μl
	\|-		\|-
	\|-		\|-
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	\|-		\|-
	! 1492R primer		! 1492R primer
-	\| 1.25	+	\| 1.25 μl
	\| 0.5 μMolar		\| 0.5 μMolar
	\|-		\|-

Janet McDonough: /* Part A: Prepare Lysates from pure cultures of 4 Bacteria of Interest */

2010-03-16T12:36:30Z

Part A: Prepare Lysates from pure cultures of 4 Bacteria of Interest

←Older revision		Revision as of 12:36, 16 March 2010
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	2. Get 4 pcr tubes of your team color (0.2ml vol) out of the jar on the instructor bench and label each with a unique code to indicate each isolate you want to id. DO NOT CONTAMINATE THE INSIDE OF THE CAP with your skin flora. Use the schematic we began with the culture independent bacterial id: your sampling habitat id (S, H, or T, including sampling site A or B if appropriate) and a unique set of numbers that your lab instructor will assign each student. Numbers found on the board or in a message to the First Class conference. If you REALLY want to send out more than 4 for id, we probably can do that. See your instructor to get approval to set up a 5th lysate. <BR><BR>		2. Get 4 pcr tubes of your team color (0.2ml vol) out of the jar on the instructor bench and label each with a unique code to indicate each isolate you want to id. DO NOT CONTAMINATE THE INSIDE OF THE CAP with your skin flora. Use the schematic we began with the culture independent bacterial id: your sampling habitat id (S, H, or T, including sampling site A or B if appropriate) and a unique set of numbers that your lab instructor will assign each student. Numbers found on the board or in a message to the First Class conference. If you REALLY want to send out more than 4 for id, we probably can do that. See your instructor to get approval to set up a 5th lysate. <BR><BR>
	3. Using your P20 micropipet, pipet 20μL of sterile water with 0.05% Non-idet P40 into each of the prelabeled pcr tubes. Nonidet-P40 is a detergent that keeps hydrophobic domains dispersed and, thus, helps to solubilize membranes. It is similar to Triton-x 100.<BR> <BR>		3. Using your P20 micropipet, pipet 20μL of sterile water with 0.05% Non-idet P40 into each of the prelabeled pcr tubes. Nonidet-P40 is a detergent that keeps hydrophobic domains dispersed and, thus, helps to solubilize membranes. It is similar to Triton-x 100.<BR> <BR>
-	4. Touch a well isolated colony from a pure culture with a P10 micropipet tip (the tiny ones, not the P20 tips) ~~or a sterile toothpick~~ and resuspend an invisible amount of bacteria adhering to the tip by swishing the tip around in the appropriately labeled tube with water and detergent. Resist the urge to pick up too much cell material but be aware that some of your isolates, particularly those dry, powdery ''Actinomycetes'', ''Steptomyces'' and the violacin producing ones are hard to get any cells to adhere. For those, it is ok to take a ''barely'' visible amount. The tinest bit is enough, but make sure there is some part of the colony going into the lysate. Putting in too much can inhibit the pcr reaction.<BR><BR>	+	4. Touch a well isolated colony from a pure culture with a sterile toothpick or a P10 micropipet tip (the tiny ones, not the P20 tips) and resuspend an invisible amount of bacteria adhering to the tip by swishing the tip around in the appropriately labeled tube with water and detergent. Resist the urge to pick up too much cell material but be aware that some of your isolates, particularly those dry, powdery ''Actinomycetes'', ''Steptomyces'' and the violacin producing ones are hard to get any cells to adhere. For those, it is ok to take a ''barely'' visible amount. The tinest bit is enough, but make sure there is some part of the colony going into the lysate. Putting in too much can inhibit the pcr reaction.<BR><BR>
	5. Repeat for your other 3 colonies into separate tubes.<BR><BR>		5. Repeat for your other 3 colonies into separate tubes.<BR><BR>
	6. Boil all 4 samples for 5 minutes. This will lyse the cells and inactivate bacterial enzymes. You can boil in a heat block or in the thermal cycler if you set a program to ''boil'' and you use the smaller pcr tubes. We will boil the tubes in the thermal cycler today so you need not worry about using the caps that prevent the tops of microfuge tubes from popping off when they are boiled. Be careful when you remove the tubes from the thermal cycler. Point them away from you and ease the lids open while still covering the outside of the tops with your gloved fingers. Do this slowly and carefully with the opening pointed away from you. You don't want the caps to pop and make an aerosol of your bacteria and you don't want to lose your lysate. <BR><BR>		6. Boil all 4 samples for 5 minutes. This will lyse the cells and inactivate bacterial enzymes. You can boil in a heat block or in the thermal cycler if you set a program to ''boil'' and you use the smaller pcr tubes. We will boil the tubes in the thermal cycler today so you need not worry about using the caps that prevent the tops of microfuge tubes from popping off when they are boiled. Be careful when you remove the tubes from the thermal cycler. Point them away from you and ease the lids open while still covering the outside of the tops with your gloved fingers. Do this slowly and carefully with the opening pointed away from you. You don't want the caps to pop and make an aerosol of your bacteria and you don't want to lose your lysate. <BR><BR>

Tucker Crum: /* Part B: PCR AMPLIFICATION of 16s rDNA from lysates prepared above */

2010-03-15T16:50:22Z

Part B: PCR AMPLIFICATION of 16s rDNA from lysates prepared above

←Older revision		Revision as of 16:50, 15 March 2010
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	The length of the fragment you are amplifying determines the extension time. A general rule of thumb is to use an extension time of 1kb per minute. Here, we amplify with primers designed for the 27th and 1492th positions in the 16s rDNA gene region. Therefore our fragment is expected to be about 1.5kb long, so we will use an extension time of 1.5 minutes per cycle. <BR><BR>		The length of the fragment you are amplifying determines the extension time. A general rule of thumb is to use an extension time of 1kb per minute. Here, we amplify with primers designed for the 27th and 1492th positions in the 16s rDNA gene region. Therefore our fragment is expected to be about 1.5kb long, so we will use an extension time of 1.5 minutes per cycle. <BR><BR>
		+
		+	'''Thermal Cycler Program:'''<br>
		+	3 step program<br>
		+
		+	{\| border="1"
		+	\|+
		+	! Cycle Step !! Temperature !! Time !! # of Cycles
		+	\|-
		+	! Initial Denaturation
		+	\| 98C
		+	\| 5 min.
		+	\| 1
		+	\|-
		+	! Denaturation <br>Annealing<br>Extension
		+	\| 98C <br> 55C<br> 72C
		+	\| 10 sec <br>30 sec <br> 30 sec
		+	\| 20
		+	\|-
		+	! Final Extension
		+	\| 72C <Br> 4C
		+	\| 10 min <BR> Hold
		+	\| 1
		+	\|-
		+	\|}
		+	<br>
		+

	The pcr will run for 45min or so. Before you go home you will load your pcr products and run a gel to assess your success at amplification of the 16S rDNA gene from each of your bacteria. Your instructor will photograph and label the gel according to the template and post the results to the conference.<BR><BR>		The pcr will run for 45min or so. Before you go home you will load your pcr products and run a gel to assess your success at amplification of the 16S rDNA gene from each of your bacteria. Your instructor will photograph and label the gel according to the template and post the results to the conference.<BR><BR>

Tucker Crum: /* Part B: PCR AMPLIFICATION of 16s rDNA from lysates prepared above */

2010-03-15T16:47:27Z

Part B: PCR AMPLIFICATION of 16s rDNA from lysates prepared above

←Older revision		Revision as of 16:47, 15 March 2010
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	==Part B: PCR AMPLIFICATION of 16s rDNA from lysates prepared above==		==Part B: PCR AMPLIFICATION of 16s rDNA from lysates prepared above==
-	'' Note: All reagents for the pcr should be kept on ice and the master mix should be thawed on ice. Since ~~Taq~~ can function at room temp, we don't want the reaction to start until all the tubes are in the thermal cycler.''<BR><BR>	+	'' Note: All reagents for the pcr should be kept on ice and the master mix should be thawed on ice. Since DNA polymerase can function at room temp, we don't want the reaction to start until all the tubes are in the thermal cycler.''<BR><BR>
	The components below have been aliquoted and prepared for you and are in pcr tubes of your team color. Label a pcr tube for each of your lysates with a fine point Sharpie on the top and side of the tube with the unique identifier for each bacterial isolate. We will set up one tube per lab as a neg control. <BR><BR>		The components below have been aliquoted and prepared for you and are in pcr tubes of your team color. Label a pcr tube for each of your lysates with a fine point Sharpie on the top and side of the tube with the unique identifier for each bacterial isolate. We will set up one tube per lab as a neg control. <BR><BR>

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	WEAR GLOVES AT ALL TIMES AND DON'T TOUCH THE INSIDE OF THE TUBE CAPS OR YOUR PIPET TIPS--Always use a new tip when going into anything in a pcr reaction. (Contamination is a significant problem in pcr)<BR><BR>		WEAR GLOVES AT ALL TIMES AND DON'T TOUCH THE INSIDE OF THE TUBE CAPS OR YOUR PIPET TIPS--Always use a new tip when going into anything in a pcr reaction. (Contamination is a significant problem in pcr)<BR><BR>

-	Using a P2 or P10 and filter tips (remember that the P2 has two red decimal place volume indicators while the P10 only has 1 red decimal place indicator. MAKE SURE YOU HAVE DIALED IN THE CORRECT VOLUME!), '''add 2 microliters of your boiled lysate''' (containing the template DNA) '''to the prealiquoted 23 microliters of master mix''' (~~containes Taq~~ polymerase, dNPTs, MgCl<sup>2</sup>, and buffers), primers and nuclease free water mixture described above (for a total volume of 25 μL) in clearly labeled pcr tubes of your team color. Make sure you label on both the top and sides of the tube. (The tubes are tiny so you will have to make an identification code and keep the key to the code in your lab notebook and give a copy to your instructor.) We need unique code names for your isolates that should continue the scheme we used for the culture-independent bacteria. For example, all isolates from the Durant Camellia house begin with S; Tropical house isolates T begin with T and isolates from around the bamboo in the Hydrophyte house begin with H. If your isolates are from sampling site A vs. B, please add that letter after the S, H, or T. If you are in the Tues lab, your isolates will start with 300 (odd numbers) and if you are in the Wed lab, your isolates start with 400 (even numbers). Each student will be assigned a unique group of numbers within that range to use for coding your isolates as S301 or H490, etc. <BR>	+	Using a P2 or P10 and filter tips (remember that the P2 has two red decimal place volume indicators while the P10 only has 1 red decimal place indicator. MAKE SURE YOU HAVE DIALED IN THE CORRECT VOLUME!), '''add 2 microliters of your boiled lysate''' (containing the template DNA) '''to the prealiquoted 23 microliters of master mix''' (contains DNA polymerase, dNPTs, MgCl<sup>2</sup>, and buffers), primers and nuclease free water mixture described above (for a total volume of 25 μL) in clearly labeled pcr tubes of your team color. Make sure you label on both the top and sides of the tube. (The tubes are tiny so you will have to make an identification code and keep the key to the code in your lab notebook and give a copy to your instructor.) We need unique code names for your isolates that should continue the scheme we used for the culture-independent bacteria. For example, all isolates from the Durant Camellia house begin with S; Tropical house isolates T begin with T and isolates from around the bamboo in the Hydrophyte house begin with H. If your isolates are from sampling site A vs. B, please add that letter after the S, H, or T. If you are in the Tues lab, your isolates will start with 300 (odd numbers) and if you are in the Wed lab, your isolates start with 400 (even numbers). Each student will be assigned a unique group of numbers within that range to use for coding your isolates as S301 or H490, etc. <BR>

-	~~Hold the tubes on ice until your instructor tells you the thermal cycler is ready to be loaded. Wipe the outside of the tubes to remove all ice and water before placing them in the thermal cycler.<BR>~~	+	'''Component TABLE '''<BR>
-		+
-	For the negative control one person will add 2 microliters of water (in place of the template DNA). When you have mixed your DNA or water into the pcr mix by tapping VERY LIGHTLY or flicking to be sure that all reagents are mixed and not adhering to the tube wall, take your tubes to the thermal cycler when your instructor says it's ready. Keep them on ice until then, but wipe off the bottom of the tubes before putting them into the machine. Make a template key in your lab notebook as to where in the thermal cycler you put your tubes. <BR><BR>	+
-		+
-	The thermal cycler program is, generally, the same for all pcr reactions, but the annealing temperature (melting) is dependent on the primer pair. When you design primers, the primer melting temp. can be calculated based on the GC content and other factors. Think about which would be harder to denature: GC pairs or AT pairs and why? For 27F and 1492R, a range of 45-55C is ok, although higher temp. may lead to increased specificity that excludes some organisms' ~~DNA from being amplified. <BR><BR>~~	+
-		+
-	The length of the fragment you are amplifying determines the extension time. A general rule of thumb is to use an extension time of 1kb per minute. Here, we amplify with primers designed for the 27th and 1492th positions in the 16s rDNA gene region. Therefore our fragment is expected to be about 1.5kb long, so we will use an extension time of 1.5 minutes per cycle. <BR><BR>	+
-		+
-	'~~''PRC Thermocycler Program= 30 cycles of:~~'''	+
	{\| border="1"		{\| border="1"
	\|+		\|+
-	! ~~Step~~ !! ~~Temp~~ !! ~~Time !~~!	+	! Component !! amt. in a 50 μl<br>reaction !! Final Conc.
		+	\|-
		+	! Purified<BR>DNAase free <BR> Water
		+	\| 13 μl
	\|-		\|-
-	~~! Initial Denaturation~~
-	~~\| 95C~~
-	~~\| 5 min.~~
	\|-		\|-
-	! ~~Denaturation of template~~	+	! 2x Phusion Master Mix
-	\| ~~95C~~	+	\| 12.5 μl
-	\| ~~30 sec.~~	+	\| 1x
	\|-		\|-
-	! ~~Annealing of primers~~	+	! 27F primer
-	\| ~~55C~~	+	\| 1.25 μl
-	\| ~~30 sec~~.	+	\| 0.5 μMolar
	\|-		\|-
-	! ~~Extension~~	+	! 1492R primer
-	\| ~~72C~~	+	\| 1.25
-	\| 1.5 ~~min.~~	+	\| 0.5 μMolar
	\|-		\|-
-	! ~~Final Extension~~	+	! template DNA
-	\| ~~72C~~	+	\| 2 μl
-	\| ~~5 min.~~	+	\| optimum is 100ng of DNA/reaction
-	\|-	+
-	~~! Hold~~	+
-	~~\| 4C~~	+
-	~~\| Forever~~	+
	\|-		\|-
	\|}		\|}
-	~~<br><BR>~~


-	~~The pcr will run for 1.5 hours or so. We can leave~~ the ~~pcr reactions in~~ the thermal cycler ~~overnight, as long as we program the last cycle~~ to be ~~''HOLD AT 4C''~~. ~~Your instructor~~ will ~~freeze away~~ your pcr ~~products tomorrow so~~ be sure ~~they~~ are ~~labeled clearly~~. ~~Next week~~ you ~~will use~~ the ~~nanodropper~~ or ~~run~~ a ~~gel~~ to ~~assess your success at amplification~~ of the ~~16S~~ rDNA gene ~~from each~~ of ~~your bacteria~~. <BR><BR>	+	Hold the tubes on ice until your instructor tells you the thermal cycler is ready to be loaded. Wipe the outside of the tubes to remove all ice and water before placing them in the thermal cycler.<BR>
		+
		+	For the negative control one person will add 2 microliters of water (in place of the template DNA). When you have mixed your DNA or water into the pcr mix by tapping VERY LIGHTLY or flicking to be sure that all reagents are mixed and not adhering to the tube wall, take your tubes to the thermal cycler when your instructor says it's ready. Keep them on ice until then, but wipe off the bottom of the tubes before putting them into the machine. Make a template key in your lab notebook as to where in the thermal cycler you put your tubes. <BR><BR>
		+
		+	The thermal cycler program is, generally, similar for all pcr reactions, but the annealing temperature (melting) is dependent on the primer pair. When you design primers, the primer melting temp. can be calculated based on the GC content and other factors. Think about which would be harder to denature: GC pairs or AT pairs and why? For 27F and 1492R, a range of 45-55C is ok, although higher temp. may lead to increased specificity that excludes some organisms' DNA from being amplified. <BR><BR>
		+
		+	The length of the fragment you are amplifying determines the extension time. A general rule of thumb is to use an extension time of 1kb per minute. Here, we amplify with primers designed for the 27th and 1492th positions in the 16s rDNA gene region. Therefore our fragment is expected to be about 1.5kb long, so we will use an extension time of 1.5 minutes per cycle. <BR><BR>

-	~~'''Master Mix recipe~~ for ~~each reaction: TOTAL VOL 23 microliters'''<BR>~~	+	The pcr will run for 45min or so. Before you go home you will load your pcr products and run a gel to assess your success at amplification of the 16S rDNA gene from each of your bacteria. Your instructor will photograph and label the gel according to the template and post the results to the conference.<BR><BR>
-	~~'''REAGENT and VOLUME'''<BR>~~	+
-	~~Promega Master Mix 2x 12~~.~~5 microliters (50 units/ml of Taq DNA polymerase supplied in~~ a ~~proprietary reaction buffer (pH 8.5, 400μM~~	+
-	~~dATP, 400μM dGTP, 400μM dCTP, 400μM dTTP, 3mM MgCl<sup>2</sup>.)<br>~~	+
-	~~16S_ 27F(15 pmol) primer 2.0 microliter<BR>~~	+
-	~~16S_1492R (15 pmol) 2.0 microliter <BR>~~	+
-	~~nuclease free water 6.5 microliter<BR>~~	+
-	~~'''23μL TOTAL reaction vol. x~~ the ~~number~~ of ~~reactions~~<BR><BR>	+

	==Part C: Culturable Bacteria Characterization by Metabolic and Physical Tests==		==Part C: Culturable Bacteria Characterization by Metabolic and Physical Tests==

Tucker Crum: /* LAB 6: Start the Characterization of Cultured Bacteria by 16srDNA Sequencing and Analysis & Continue Traditional ID Techniques */

2010-03-15T16:35:04Z

LAB 6: Start the Characterization of Cultured Bacteria by 16srDNA Sequencing and Analysis & Continue Traditional ID Techniques

←Older revision		Revision as of 16:35, 15 March 2010
Line 6:		Line 6:
	In addition to isolating genomic DNA from a soil sample, amplifying the 16S rDNA by pcr, inserting the different DNA fragments from the pcr product into a cloning vector, transforming ''E. coli'' with your clones, and sending the transformed ''E. coli'' off to have the 16s rDNA inserts sequenced in an automatic sequencer so that you can identify a large and, we hope, representative sample of the bacterial flora in the soil community from your habitat, you also have been working, simultaneously, through traditional microbiological culturing techniques, to isolate and characterize a few of the culturable bacteria through morphology, physical, and metabolism differentiation. Look how much you have accomplished in these few short weeks!!<br><BR>		In addition to isolating genomic DNA from a soil sample, amplifying the 16S rDNA by pcr, inserting the different DNA fragments from the pcr product into a cloning vector, transforming ''E. coli'' with your clones, and sending the transformed ''E. coli'' off to have the 16s rDNA inserts sequenced in an automatic sequencer so that you can identify a large and, we hope, representative sample of the bacterial flora in the soil community from your habitat, you also have been working, simultaneously, through traditional microbiological culturing techniques, to isolate and characterize a few of the culturable bacteria through morphology, physical, and metabolism differentiation. Look how much you have accomplished in these few short weeks!!<br><BR>

-	By this point you have isolated pure colonies of some soil bacteria on general and enrichment media and you have gotten some preliminary or defining information about the morphologic and metabolic characteristic of the bacteria you have chosen to identify. You will continue learning about how these bacteria are different from one another and how they contribute to their community through research and performing more tests. At the same time we want to identify these bacteria by their 16s rDNA unique sequences. The process will be somewhat simplier this time. We don't have to clone into a vector and transform bacteria. This time we will use a ~~Taq~~ polymerase, ~~rather than a~~ proof-reading DNA polymerase. ~~Taq will not be as accurate as our proof reading polymerase, but it is much less expensive and should be good enough to allow us to get genus and species identification from DNA sequencing.<BR><BR>~~ In schematic the process goes as follows:<BR>	+	By this point you have isolated pure colonies of some soil bacteria on general and enrichment media and you have gotten some preliminary or defining information about the morphologic and metabolic characteristic of the bacteria you have chosen to identify. You will continue learning about how these bacteria are different from one another and how they contribute to their community through research and performing more tests. At the same time we want to identify these bacteria by their 16s rDNA unique sequences. The process will be somewhat simplier this time. We don't have to clone into a vector and transform bacteria. This time we will use a the Finnzyme Phusion™ polymerase, the proof-reading DNA polymerase. In schematic the process goes as follows:<BR>
	<center><font color="blue">'''To Identify Bacteria from DNA from Isolated Pure Colonies'''</font color="blue"></center>		<center><font color="blue">'''To Identify Bacteria from DNA from Isolated Pure Colonies'''</font color="blue"></center>
	<br>		<br>

Tucker Crum: /* Part A: Prepare Lysates from pure cultures of 4 Bacteria of Interest */

2010-03-15T16:31:56Z

Part A: Prepare Lysates from pure cultures of 4 Bacteria of Interest

←Older revision		Revision as of 16:31, 15 March 2010
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	2. Get 4 pcr tubes of your team color (0.2ml vol) out of the jar on the instructor bench and label each with a unique code to indicate each isolate you want to id. DO NOT CONTAMINATE THE INSIDE OF THE CAP with your skin flora. Use the schematic we began with the culture independent bacterial id: your sampling habitat id (S, H, or T, including sampling site A or B if appropriate) and a unique set of numbers that your lab instructor will assign each student. Numbers found on the board or in a message to the First Class conference. If you REALLY want to send out more than 4 for id, we probably can do that. See your instructor to get approval to set up a 5th lysate. <BR><BR>		2. Get 4 pcr tubes of your team color (0.2ml vol) out of the jar on the instructor bench and label each with a unique code to indicate each isolate you want to id. DO NOT CONTAMINATE THE INSIDE OF THE CAP with your skin flora. Use the schematic we began with the culture independent bacterial id: your sampling habitat id (S, H, or T, including sampling site A or B if appropriate) and a unique set of numbers that your lab instructor will assign each student. Numbers found on the board or in a message to the First Class conference. If you REALLY want to send out more than 4 for id, we probably can do that. See your instructor to get approval to set up a 5th lysate. <BR><BR>
	3. Using your P20 micropipet, pipet 20μL of sterile water with 0.05% Non-idet P40 into each of the prelabeled pcr tubes. Nonidet-P40 is a detergent that keeps hydrophobic domains dispersed and, thus, helps to solubilize membranes. It is similar to Triton-x 100.<BR> <BR>		3. Using your P20 micropipet, pipet 20μL of sterile water with 0.05% Non-idet P40 into each of the prelabeled pcr tubes. Nonidet-P40 is a detergent that keeps hydrophobic domains dispersed and, thus, helps to solubilize membranes. It is similar to Triton-x 100.<BR> <BR>
-	4. Touch a well isolated colony from a pure culture with a P10 micropipet tip (the tiny ones, not the P20 tips) and resuspend ~~a tiny, barely visible~~ amount of bacteria adhering to the tip by swishing the tip around in the appropriately labeled tube with water and detergent. Resist the urge to pick up too much cell material but be aware that some of your isolates, particularly those dry, powdery ''Actinomycetes'', ''Steptomyces'' and the violacin producing ones are hard to get any cells to adhere. ~~Make~~ sure there is some part of the colony going into the lysate~~. The tinest bit is enough~~. Putting in too much can inhibit the pcr reaction.<BR><BR>	+	4. Touch a well isolated colony from a pure culture with a P10 micropipet tip (the tiny ones, not the P20 tips) or a sterile toothpick and resuspend an invisible amount of bacteria adhering to the tip by swishing the tip around in the appropriately labeled tube with water and detergent. Resist the urge to pick up too much cell material but be aware that some of your isolates, particularly those dry, powdery ''Actinomycetes'', ''Steptomyces'' and the violacin producing ones are hard to get any cells to adhere. For those, it is ok to take a ''barely'' visible amount. The tinest bit is enough, but make sure there is some part of the colony going into the lysate. Putting in too much can inhibit the pcr reaction.<BR><BR>
	5. Repeat for your other 3 colonies into separate tubes.<BR><BR>		5. Repeat for your other 3 colonies into separate tubes.<BR><BR>
	6. Boil all 4 samples for 5 minutes. This will lyse the cells and inactivate bacterial enzymes. You can boil in a heat block or in the thermal cycler if you set a program to ''boil'' and you use the smaller pcr tubes. We will boil the tubes in the thermal cycler today so you need not worry about using the caps that prevent the tops of microfuge tubes from popping off when they are boiled. Be careful when you remove the tubes from the thermal cycler. Point them away from you and ease the lids open while still covering the outside of the tops with your gloved fingers. Do this slowly and carefully with the opening pointed away from you. You don't want the caps to pop and make an aerosol of your bacteria and you don't want to lose your lysate. <BR><BR>		6. Boil all 4 samples for 5 minutes. This will lyse the cells and inactivate bacterial enzymes. You can boil in a heat block or in the thermal cycler if you set a program to ''boil'' and you use the smaller pcr tubes. We will boil the tubes in the thermal cycler today so you need not worry about using the caps that prevent the tops of microfuge tubes from popping off when they are boiled. Be careful when you remove the tubes from the thermal cycler. Point them away from you and ease the lids open while still covering the outside of the tops with your gloved fingers. Do this slowly and carefully with the opening pointed away from you. You don't want the caps to pop and make an aerosol of your bacteria and you don't want to lose your lysate. <BR><BR>