BISC209: Lab3 - Revision history

Tucker Crum: /* Part B: PCR Amplification of 16s rRNA genes from Universal Bacterial Primers */

Tucker Crum — Wed, 17 Mar 2010 12:12:20 GMT

Part B: PCR Amplification of 16s rRNA genes from Universal Bacterial Primers

←Older revision		Revision as of 12:12, 17 March 2010
Line 74:		Line 74:
	Label it with a fine tipped Sharpie on the top and side with the code name for your sample. Do not use tape. <BR><BR>		Label it with a fine tipped Sharpie on the top and side with the code name for your sample. Do not use tape. <BR><BR>

-	'''If your soil DNA isolate is at approximately 100ng/μL, you will follow the Template Table (shown below) adding ~~4μL of~~ DNAase free water and only 1μL of template DNA to the reagents that have already been premixed for you in your pcr tube (~~12.5μL~~ master mix, ~~5μL~~ DNAase free water, ~~1.25μL~~ of each of 2 primers).'''<BR><BR>	+	'''If your soil DNA isolate is at approximately 100ng/μL, you will follow the Template Table (shown below) adding 3μL DNAase free water and only 1μL of template DNA to the reagents that have already been premixed for you in your pcr tube (10μL master mix, 4μL DNAase free water, 1μL of each of 2 primers).'''<BR><BR>

-	'''If your soil isolate DNA concentration was less than 20ng/μL, you will add 5 μL of DNA and no extra water. If your concentration was between 20 and 100ng/μL, calculate how much template DNA to add by using the formula 100 / your isolate's DNA conc. Add that number of microliters of DNA (not more than 5) and enough DNAase free water so that the number of microliters of DNA + microliters of water =5. Example: Your DNA conc. was 33ng/μL. 100/33 = 3.3 so you would add 3.3μL of DNA and 1.7μL of DNAase free water. Since your pcr tube already has ~~12.5μL~~ master mix, ~~5μL~~ DNAase free water, and ~~1.25μL~~ of each of 2 primers, the total reaction volume for everyone will be ~~25μL~~.'''<BR><BR>	+	'''If your soil isolate DNA concentration was less than 20ng/μL, you will add 4 μL of DNA and no extra water. If your concentration was between 20 and 100ng/μL, calculate how much template DNA to add by using the formula 100 / your isolate's DNA conc. Add that number of microliters of DNA (not more than 4) and enough DNAase free water so that the number of microliters of DNA + microliters of water =4. Example: Your DNA conc. was 33ng/μL. 100/33 = 3.3 so you would add 3.3μL of DNA and 0.7μL of DNAase free water. Since your pcr tube already has 10μL master mix, 4μL DNAase free water, and 1μL of each of 2 primers, the total reaction volume for everyone will be 20μL.'''<BR><BR>

	It is very important to pipet these tiny volumes accurately. Use a P2 or P10 and the special small tips with a filter when pipetting DNA. Look at the tip after you draw up your measured volume to make sure you have liquid there. <BR><BR>		It is very important to pipet these tiny volumes accurately. Use a P2 or P10 and the special small tips with a filter when pipetting DNA. Look at the tip after you draw up your measured volume to make sure you have liquid there. <BR><BR>
Line 86:		Line 86:
	{\| border="1"		{\| border="1"
	\|+		\|+
-	! Component !! amt. in a 25 μl<br>reaction !! Final Conc.	+	! Component !! amt. in a 20 μl<br>reaction !! Final Conc.
	\|-		\|-
	! Purified<BR>DNAase free <BR> Water		! Purified<BR>DNAase free <BR> Water
-	\| 5 μL already in tube.<BR> Want to achieve<br>total of 25 μl reaction vol.<br> Add from 0 - 4μl	+	\| 4 μL already in tube.<BR> Want to achieve<br>total of 20 μl reaction vol.<br> Add from 0 - 4μl
	\| _		\| _
	\|-		\|-
	! 2x Phusion Master Mix		! 2x Phusion Master Mix
-	\| ~~12.5~~ μl	+	\| 10 μl
	\| 1x		\| 1x
	\|-		\|-
	! 27F primer		! 27F primer
-	\| 1~~.25~~	+	\| 1
	\| 0.5 μMolar		\| 0.5 μMolar
	\|-		\|-
	! 1492R primer		! 1492R primer
-	\| 1~~.25~~	+	\| 1
	\| 0.5 μMolar		\| 0.5 μMolar
	\|-		\|-
	! template DNA		! template DNA
-	\| 1-5 μl	+	\| 1-4 μl
	\| optimum is 100ng of DNA/reaction		\| optimum is 100ng of DNA/reaction
	\|-		\|-

Tucker Crum: /* Part B: PCR Amplification of 16s rRNA genes from Universal Bacterial Primers */

Tucker Crum — Wed, 17 Mar 2010 11:47:35 GMT

Part B: PCR Amplification of 16s rRNA genes from Universal Bacterial Primers

←Older revision		Revision as of 11:47, 17 March 2010
Line 74:		Line 74:
	Label it with a fine tipped Sharpie on the top and side with the code name for your sample. Do not use tape. <BR><BR>		Label it with a fine tipped Sharpie on the top and side with the code name for your sample. Do not use tape. <BR><BR>

-	'''If your soil DNA isolate is at approximately 100ng/μL, you will follow the Template Table (shown below) adding 4μL of DNAase free water and only 1μL of template DNA to the reagents that have already been premixed for you in your pcr tube (~~25μL~~ master mix, ~~19μL~~ DNAase free water, 2.~~5μL~~ of each of 2 primers).'''<BR><BR>	+	'''If your soil DNA isolate is at approximately 100ng/μL, you will follow the Template Table (shown below) adding 4μL of DNAase free water and only 1μL of template DNA to the reagents that have already been premixed for you in your pcr tube (12.5μL master mix, 5μL DNAase free water, 1.25μL of each of 2 primers).'''<BR><BR>

-	'''If your soil isolate DNA concentration was less than 20ng/μL, you will add 5 μL of DNA and no extra water. If your concentration was between 20 and 100ng/μL, calculate how much template DNA to add by using the formula 100 / your isolate's DNA conc. Add that number of microliters of DNA (not more than 5) and enough DNAase free water so that the number of microliters of DNA + microliters of water =5. Example: Your DNA conc. was 33ng/μL. 100/33 = 3.3 so you would add 3.3μL of DNA and 1.7μL of DNAase free water. Since your pcr tube already has ~~25μL~~ master mix, ~~19μL~~ DNAase free water, and 2.~~5μL~~ of each of 2 primers, the total reaction volume for everyone will be ~~50μL~~.'''<BR><BR>	+	'''If your soil isolate DNA concentration was less than 20ng/μL, you will add 5 μL of DNA and no extra water. If your concentration was between 20 and 100ng/μL, calculate how much template DNA to add by using the formula 100 / your isolate's DNA conc. Add that number of microliters of DNA (not more than 5) and enough DNAase free water so that the number of microliters of DNA + microliters of water =5. Example: Your DNA conc. was 33ng/μL. 100/33 = 3.3 so you would add 3.3μL of DNA and 1.7μL of DNAase free water. Since your pcr tube already has 12.5μL master mix, 5μL DNAase free water, and 1.25μL of each of 2 primers, the total reaction volume for everyone will be 25μL.'''<BR><BR>

	It is very important to pipet these tiny volumes accurately. Use a P2 or P10 and the special small tips with a filter when pipetting DNA. Look at the tip after you draw up your measured volume to make sure you have liquid there. <BR><BR>		It is very important to pipet these tiny volumes accurately. Use a P2 or P10 and the special small tips with a filter when pipetting DNA. Look at the tip after you draw up your measured volume to make sure you have liquid there. <BR><BR>
Line 86:		Line 86:
	{\| 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
-	\| 19 μL already in tube.<BR> Want to achieve<br>total of 50 μl reaction vol.<br> Add from 0 - 4μl	+	\| 5 μL already in tube.<BR> Want to achieve<br>total of 25 μl reaction vol.<br> Add from 0 - 4μl
	\| _		\| _
	\|-		\|-
	! 2x Phusion Master Mix		! 2x Phusion Master Mix
-	\| 25 μl	+	\| 12.5 μl
	\| 1x		\| 1x
	\|-		\|-
	! 27F primer		! 27F primer
-	\| 2.5	+	\| 1.25
	\| 0.5 μMolar		\| 0.5 μMolar
	\|-		\|-
	! 1492R primer		! 1492R primer
-	\| 2.5	+	\| 1.25
	\| 0.5 μMolar		\| 0.5 μMolar
	\|-		\|-

Tucker Crum: /* Part B: PCR Amplification of 16s rRNA genes from Universal Bacterial Primers */

Tucker Crum — Mon, 15 Mar 2010 16:36:44 GMT

Part B: PCR Amplification of 16s rRNA genes from Universal Bacterial Primers

←Older revision		Revision as of 16:36, 15 March 2010
Line 83:		Line 83:
	Tap the bottom of the tube (VERY GENTLY!) and flick the tube to mix. Do not treat these tubes roughly as they are quite thin-walled and can break or crack. <BR><BR>		Tap the bottom of the tube (VERY GENTLY!) and flick the tube to mix. Do not treat these tubes roughly as they are quite thin-walled and can break or crack. <BR><BR>
	Bring your tube to your instructor; she will show you where the thermal cycler is located in E301. Your instructor will start the reaction when everyone's tubes are loaded. <BR><BR>		Bring your tube to your instructor; she will show you where the thermal cycler is located in E301. Your instructor will start the reaction when everyone's tubes are loaded. <BR><BR>
-	'''~~TEMPLATE~~ TABLE '''<BR>	+	'''Component TABLE '''<BR>
	{\| border="1"		{\| border="1"
	\|+		\|+

Tucker Crum: /* Part C: Clean Up of pcr product using Epoch BIoLabs GenCatch PCR CleanUp Kit */

Tucker Crum — Thu, 11 Mar 2010 13:52:27 GMT

Part C: Clean Up of pcr product using Epoch BIoLabs GenCatch PCR CleanUp Kit

←Older revision		Revision as of 13:52, 11 March 2010
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	agent. ''<BR><BR>		agent. ''<BR><BR>

-	==~~'''~~Agarose Gel Electrophoresis of Clean PCR PRODUCT~~'''~~==~~<BR>~~	+	==Agarose Gel Electrophoresis of Clean PCR PRODUCT==
	To see if you successfully amplified the 16s rRNA gene and not anything else, you will "run a gel" on your cleaned pcr products. To run a gel means that we will perform an electrophoretic separation of the DNA fragments in your cleaned up pcr product, using 1/10 vol. of your pcr product applied to a 1% agarose gel stained with Sybr Safe DNA stain. Your instructor will photograph the gel, label it with your amplicon id from the template and post the gel photo to the data folder in the First Class lab conference so you can evaluate your success at 16S rRNA gene amplification. You should see a single band of ~1.5kb indicating that the only dsDNA in your pcr product came from amplification of a ~1500bp gene fragment. Can you explain how we know the size of our amplified gene fragment?<BR><BR>		To see if you successfully amplified the 16s rRNA gene and not anything else, you will "run a gel" on your cleaned pcr products. To run a gel means that we will perform an electrophoretic separation of the DNA fragments in your cleaned up pcr product, using 1/10 vol. of your pcr product applied to a 1% agarose gel stained with Sybr Safe DNA stain. Your instructor will photograph the gel, label it with your amplicon id from the template and post the gel photo to the data folder in the First Class lab conference so you can evaluate your success at 16S rRNA gene amplification. You should see a single band of ~1.5kb indicating that the only dsDNA in your pcr product came from amplification of a ~1500bp gene fragment. Can you explain how we know the size of our amplified gene fragment?<BR><BR>

Tucker Crum: /* Part C: Clean Up of pcr product using Epoch BIoLabs GenCatch PCR CleanUp Kit */

Tucker Crum — Thu, 11 Mar 2010 13:51:38 GMT

Part C: Clean Up of pcr product using Epoch BIoLabs GenCatch PCR CleanUp Kit

←Older revision		Revision as of 13:51, 11 March 2010
Line 163:		Line 163:
	agent. ''<BR><BR>		agent. ''<BR><BR>

-	'''Agarose Gel Electrophoresis'''<BR>	+	=='''Agarose Gel Electrophoresis of Clean PCR PRODUCT'''==<BR>
	To see if you successfully amplified the 16s rRNA gene and not anything else, you will "run a gel" on your cleaned pcr products. To run a gel means that we will perform an electrophoretic separation of the DNA fragments in your cleaned up pcr product, using 1/10 vol. of your pcr product applied to a 1% agarose gel stained with Sybr Safe DNA stain. Your instructor will photograph the gel, label it with your amplicon id from the template and post the gel photo to the data folder in the First Class lab conference so you can evaluate your success at 16S rRNA gene amplification. You should see a single band of ~1.5kb indicating that the only dsDNA in your pcr product came from amplification of a ~1500bp gene fragment. Can you explain how we know the size of our amplified gene fragment?<BR><BR>		To see if you successfully amplified the 16s rRNA gene and not anything else, you will "run a gel" on your cleaned pcr products. To run a gel means that we will perform an electrophoretic separation of the DNA fragments in your cleaned up pcr product, using 1/10 vol. of your pcr product applied to a 1% agarose gel stained with Sybr Safe DNA stain. Your instructor will photograph the gel, label it with your amplicon id from the template and post the gel photo to the data folder in the First Class lab conference so you can evaluate your success at 16S rRNA gene amplification. You should see a single band of ~1.5kb indicating that the only dsDNA in your pcr product came from amplification of a ~1500bp gene fragment. Can you explain how we know the size of our amplified gene fragment?<BR><BR>

Tucker Crum: /* Agarose Gel Electrophoresis of Cleaned PCR Products */

Tucker Crum — Thu, 11 Mar 2010 13:50:49 GMT

Agarose Gel Electrophoresis of Cleaned PCR Products

←Older revision		Revision as of 13:50, 11 March 2010
Line 163:		Line 163:
	agent. ''<BR><BR>		agent. ''<BR><BR>

-	==Agarose Gel Electrophoresis ~~of Cleaned PCR Products==~~	+	'''Agarose Gel Electrophoresis'''<BR>
	To see if you successfully amplified the 16s rRNA gene and not anything else, you will "run a gel" on your cleaned pcr products. To run a gel means that we will perform an electrophoretic separation of the DNA fragments in your cleaned up pcr product, using 1/10 vol. of your pcr product applied to a 1% agarose gel stained with Sybr Safe DNA stain. Your instructor will photograph the gel, label it with your amplicon id from the template and post the gel photo to the data folder in the First Class lab conference so you can evaluate your success at 16S rRNA gene amplification. You should see a single band of ~1.5kb indicating that the only dsDNA in your pcr product came from amplification of a ~1500bp gene fragment. Can you explain how we know the size of our amplified gene fragment?<BR><BR>		To see if you successfully amplified the 16s rRNA gene and not anything else, you will "run a gel" on your cleaned pcr products. To run a gel means that we will perform an electrophoretic separation of the DNA fragments in your cleaned up pcr product, using 1/10 vol. of your pcr product applied to a 1% agarose gel stained with Sybr Safe DNA stain. Your instructor will photograph the gel, label it with your amplicon id from the template and post the gel photo to the data folder in the First Class lab conference so you can evaluate your success at 16S rRNA gene amplification. You should see a single band of ~1.5kb indicating that the only dsDNA in your pcr product came from amplification of a ~1500bp gene fragment. Can you explain how we know the size of our amplified gene fragment?<BR><BR>
		+
		+	Your agarose gel is made of 1.0% agarose solution (w/v) in 1x TGE buffer (10x=0.25 Tris, 1.9M Glycine, 13mM EDTA) with SybrSafe™ stain.<BR><BR>
		+
		+	DNA is uniformly negatively charged and will,therefore, move toward the positive electrode. The separation is determined by the size or mass of the molecule or fragments of DNA. <BR><BR>
		+	[[Image:BISC110_gel2.jpg]]<BR><BR>
		+

	''' Procedure for Agarose Gel Electrophoresis of PCR products'''<BR>		''' Procedure for Agarose Gel Electrophoresis of PCR products'''<BR>
	Load 1/10 of the total volume of pcr product (1 microliter minimum). In our case we should load 5 microliters.<BR>		Load 1/10 of the total volume of pcr product (1 microliter minimum). In our case we should load 5 microliters.<BR>
-	You will put the 5 microliters of your pcr product as a spot on a small piece of parafilm and add ~~2.5microliters~~ of loading dye (0.25% XC, 30% glycerol, 0.1mg/ml RNAase). Mix the ~~solution~~ by pipetting up and down before	+	You will put the 5 microliters of your pcr product as a spot on a small piece of parafilm and add 5 microliters of loading dye (0.25% XC, 30% glycerol, 0.1mg/ml RNAase). '''Mix the loading dye''' by pipetting up and down before loading all 10 microliters into a lane of the 1% agarose gel (1% wt/vol in 1xTGE buffer with Sybr Safe DNA stain a proprietary reagent from Invitrogen used according to manufacturer's directions http://www.invitrogen.com) that you have signed up for on the gel template. Be sure to leave the first two lanes and the last lane empty for the 100bp ladder, the positive control and the negative water control.<BR><BR>
-	loading all ~~7.5~~ microliters into a lane of the 1% agarose gel (1% wt/vol in 1xTGE buffer with Sybr Safe DNA stain a proprietary reagent from Invitrogen used according to manufacturer's directions http://www.invitrogen.com) that you have signed up for on the gel template. Be sure to leave the first two lanes and the last lane empty for the 100bp ladder, the positive control and the negative water control.<BR><BR>	+
	Loading dye contains glycerol to keep our sample in the lane rather than floating away and will have one of 3 marker dyes (bromophenol blue, xylene cyanol, or		Loading dye contains glycerol to keep our sample in the lane rather than floating away and will have one of 3 marker dyes (bromophenol blue, xylene cyanol, or
	orange G) that facilitate estimation of DNA migration distance and optimization		orange G) that facilitate estimation of DNA migration distance and optimization
	of agarose gel run time. 1x TGE buffer is used in this electrophoretic separation (25mM Tris, 0.19M glycine, 1.3mM EDTA. The gel will be run at 120V for approximately 30 minutes. <BR><BR>		of agarose gel run time. 1x TGE buffer is used in this electrophoretic separation (25mM Tris, 0.19M glycine, 1.3mM EDTA. The gel will be run at 120V for approximately 30 minutes. <BR><BR>
		+
		+	How will you judge a successful amplification? How many fragments and of what size do you expect to see? <BR><BR>

	Make sure you give back the rest of your soil DNA isolate and the rest of the cleaned up pcr product to your instructor to freeze after the gel is loaded. Both are now in identical looking microfuge tubes with volume being the only visible difference. Make sure it is clear which is the pcr product and which is the genomic DNA isolate. <BR><BR>		Make sure you give back the rest of your soil DNA isolate and the rest of the cleaned up pcr product to your instructor to freeze after the gel is loaded. Both are now in identical looking microfuge tubes with volume being the only visible difference. Make sure it is clear which is the pcr product and which is the genomic DNA isolate. <BR><BR>

Tucker Crum: /* Agarose Gel Electrophoresis of Cleaned PCR Products */

Tucker Crum — Wed, 24 Feb 2010 15:58:47 GMT

Agarose Gel Electrophoresis of Cleaned PCR Products

←Older revision		Revision as of 15:58, 24 February 2010
Line 169:		Line 169:
	Load 1/10 of the total volume of pcr product (1 microliter minimum). In our case we should load 5 microliters.<BR>		Load 1/10 of the total volume of pcr product (1 microliter minimum). In our case we should load 5 microliters.<BR>
	You will put the 5 microliters of your pcr product as a spot on a small piece of parafilm and add 2.5microliters of loading dye (0.25% XC, 30% glycerol, 0.1mg/ml RNAase). Mix the solution by pipetting up and down before		You will put the 5 microliters of your pcr product as a spot on a small piece of parafilm and add 2.5microliters of loading dye (0.25% XC, 30% glycerol, 0.1mg/ml RNAase). Mix the solution by pipetting up and down before
-	loading all 7.5 microliters into a lane of the 1% agarose gel (1% wt/vol in 1xTGE buffer)that you have signed up for on the gel template. Be sure to leave the first two lanes and the last lane empty for the 100bp ladder, the positive control and the negative water control.<BR><BR>	+	loading all 7.5 microliters into a lane of the 1% agarose gel (1% wt/vol in 1xTGE buffer with Sybr Safe DNA stain a proprietary reagent from Invitrogen used according to manufacturer's directions http://www.invitrogen.com) that you have signed up for on the gel template. Be sure to leave the first two lanes and the last lane empty for the 100bp ladder, the positive control and the negative water control.<BR><BR>
	Loading dye contains glycerol to keep our sample in the lane rather than floating away and will have one of 3 marker dyes (bromophenol blue, xylene cyanol, or		Loading dye contains glycerol to keep our sample in the lane rather than floating away and will have one of 3 marker dyes (bromophenol blue, xylene cyanol, or
	orange G) that facilitate estimation of DNA migration distance and optimization		orange G) that facilitate estimation of DNA migration distance and optimization
-	of agarose gel run time. 1x TGE buffer is used in this electrophoretic separation (25mM Tris, 0.19M glycine, 1.3mM EDTA)<BR><BR>	+	of agarose gel run time. 1x TGE buffer is used in this electrophoretic separation (25mM Tris, 0.19M glycine, 1.3mM EDTA. The gel will be run at 120V for approximately 30 minutes. <BR><BR>

	Make sure you give back the rest of your soil DNA isolate and the rest of the cleaned up pcr product to your instructor to freeze after the gel is loaded. Both are now in identical looking microfuge tubes with volume being the only visible difference. Make sure it is clear which is the pcr product and which is the genomic DNA isolate. <BR><BR>		Make sure you give back the rest of your soil DNA isolate and the rest of the cleaned up pcr product to your instructor to freeze after the gel is loaded. Both are now in identical looking microfuge tubes with volume being the only visible difference. Make sure it is clear which is the pcr product and which is the genomic DNA isolate. <BR><BR>

Tucker Crum: /* Agarose Gel Electrophoresis of Cleaned PCR Products */

Tucker Crum — Wed, 24 Feb 2010 15:56:29 GMT

Agarose Gel Electrophoresis of Cleaned PCR Products

←Older revision		Revision as of 15:56, 24 February 2010
Line 168:		Line 168:
	''' Procedure for Agarose Gel Electrophoresis of PCR products'''<BR>		''' Procedure for Agarose Gel Electrophoresis of PCR products'''<BR>
	Load 1/10 of the total volume of pcr product (1 microliter minimum). In our case we should load 5 microliters.<BR>		Load 1/10 of the total volume of pcr product (1 microliter minimum). In our case we should load 5 microliters.<BR>
-	You will put the 5 microliters of your pcr product as a spot on a small piece of parafilm and add 2.5microliters of loading dye. Mix the solution by pipetting up and down before	+	You will put the 5 microliters of your pcr product as a spot on a small piece of parafilm and add 2.5microliters of loading dye (0.25% XC, 30% glycerol, 0.1mg/ml RNAase). Mix the solution by pipetting up and down before
-	loading all 7.5 microliters into a lane of the gel that you have signed up for on the gel template. Be sure to leave the first two lanes and the last lane empty for the 100bp ladder, the positive control and the negative water control.<BR><BR>	+	loading all 7.5 microliters into a lane of the 1% agarose gel (1% wt/vol in 1xTGE buffer)that you have signed up for on the gel template. Be sure to leave the first two lanes and the last lane empty for the 100bp ladder, the positive control and the negative water control.<BR><BR>
	Loading dye contains glycerol to keep our sample in the lane rather than floating away and will have one of 3 marker dyes (bromophenol blue, xylene cyanol, or		Loading dye contains glycerol to keep our sample in the lane rather than floating away and will have one of 3 marker dyes (bromophenol blue, xylene cyanol, or
	orange G) that facilitate estimation of DNA migration distance and optimization		orange G) that facilitate estimation of DNA migration distance and optimization
-	of agarose gel run time. <BR><BR>	+	of agarose gel run time. 1x TGE buffer is used in this electrophoretic separation (25mM Tris, 0.19M glycine, 1.3mM EDTA)<BR><BR>

	Make sure you give back the rest of your soil DNA isolate and the rest of the cleaned up pcr product to your instructor to freeze after the gel is loaded. Both are now in identical looking microfuge tubes with volume being the only visible difference. Make sure it is clear which is the pcr product and which is the genomic DNA isolate. <BR><BR>		Make sure you give back the rest of your soil DNA isolate and the rest of the cleaned up pcr product to your instructor to freeze after the gel is loaded. Both are now in identical looking microfuge tubes with volume being the only visible difference. Make sure it is clear which is the pcr product and which is the genomic DNA isolate. <BR><BR>

Tucker Crum: /* Part D: Finishing the Standard Plate Count of Culturable Soil Microbial Flora & Observing Colony Morphology in the Streak Plates */

Tucker Crum — Wed, 10 Feb 2010 20:31:19 GMT

Part D: Finishing the Standard Plate Count of Culturable Soil Microbial Flora & Observing Colony Morphology in the Streak Plates

←Older revision		Revision as of 20:31, 10 February 2010
Line 190:		Line 190:

	for example if you counted 150 colonies on the 10<sup>-6</sup> plate the calculation is: <BR>		for example if you counted 150 colonies on the 10<sup>-6</sup> plate the calculation is: <BR>
-	150/0.1ml plated*1X10<sup>-6</sup>dilution= 150X10<sup>7</sup> which in scientific notation is written as 1.5X10<sup>9</sup> CFU/gram <BR>	+	150/(0.1ml plated*1X10<sup>-6</sup>dilution)= 150X10<sup>7</sup> which in scientific notation is written as 1.5X10<sup>9</sup> CFU/gram <BR>

	Soil bacteria are usually, however, recorded as number of colony forming units in 1 gram of soil DRY weight. Therefore, you need to weigh each of the three 1 gram samples that you left for oven drying and average the weights. The weights should be considerably less than 1 gram. <BR>		Soil bacteria are usually, however, recorded as number of colony forming units in 1 gram of soil DRY weight. Therefore, you need to weigh each of the three 1 gram samples that you left for oven drying and average the weights. The weights should be considerably less than 1 gram. <BR>

Tucker Crum: /* Agarose Gel Electrophoresis of Cleaned PCR Products */

Tucker Crum — Wed, 10 Feb 2010 16:02:33 GMT

Agarose Gel Electrophoresis of Cleaned PCR Products

←Older revision		Revision as of 16:02, 10 February 2010
Line 174:		Line 174:
	of agarose gel run time. <BR><BR>		of agarose gel run time. <BR><BR>

-	~~Give~~ the rest of the cleaned up pcr product to your instructor to freeze after the gel is loaded~~, making~~ sure your pcr product is clearly labeled ~~with~~ your initials, team color, lab section (Tues or Wed), soil identifier code (T[tropical], CT[cool temperate], WT[warm temperate] and sample A or B (if there are two samples for a habitat) and the conc. of the DNA in ng/μL. In the next lab you will clone the fragments of 16s rDNA from the soil bacteria flora that are in your pcr product into a special genetically engineered cloning vector. That vector will be transformed into competent genetically engineered ''E. coli'' bacteria and they will be plated on selective media to find cells containing the 16s rDNA insert that we can send away for sequence analysis to determine the identity of some of the bacterial flora in your original soil sample. <BR><BR>	+	Make sure you give back the rest of your soil DNA isolate and the rest of the cleaned up pcr product to your instructor to freeze after the gel is loaded. Both are now in identical looking microfuge tubes with volume being the only visible difference. Make sure it is clear which is the pcr product and which is the genomic DNA isolate. <BR><BR>
		+
		+	Make sure your pcr product is clearly labeled as pcr product and has your initials, team color, lab section (Tues or Wed), soil identifier code (T[tropical], CT[cool temperate], WT[warm temperate] and sample A or B (if there are two samples for a habitat) and the conc. of the DNA in ng/μL. In the next lab you will clone the fragments of 16s rDNA from the soil bacteria flora that are in your pcr product into a special genetically engineered cloning vector. That vector will be transformed into competent genetically engineered ''E. coli'' bacteria and they will be plated on selective media to find cells containing the 16s rDNA insert that we can send away for sequence analysis to determine the identity of some of the bacterial flora in your original soil sample. <BR><BR>

	==Isolation & Characterization of Cultured Bacteria from a Soil Habitat==		==Isolation & Characterization of Cultured Bacteria from a Soil Habitat==