BISC209/S11: Lab4 - Revision history

Tucker Crum: /* Assignment */

Tucker Crum — Fri, 18 Feb 2011 13:03:36 GMT

Assignment

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	This assignment is due at the BEGINNING of Lab 5. Do not come late to lab because you are printing or otherwise completing this assignment and you may NOT work on it during lab. There is a 5% per day late penalty for work for this course and since you have a week or more to complete assignments, illness (unless it is lengthy and serious) does not excuse you from the late penalty.<BR><BR>		This assignment is due at the BEGINNING of Lab 5. Do not come late to lab because you are printing or otherwise completing this assignment and you may NOT work on it during lab. There is a 5% per day late penalty for work for this course and since you have a week or more to complete assignments, illness (unless it is lengthy and serious) does not excuse you from the late penalty.<BR><BR>

-	'''Continue monitoring and following the appropriate protocols to isolate our targeted ~~culturable~~ bacteria.'''	+	'''Continue monitoring and following the appropriate protocols to isolate to pure culture our targeted bacteria.'''

	==Links to Labs==		==Links to Labs==

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

Tucker Crum — Fri, 18 Feb 2011 13:02:47 GMT

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

←Older revision		Revision as of 13:02, 18 February 2011
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	==Part B: Clean Up of pcr product using Epoch BIoLabs GenCatch PCR CleanUp Kit==		==Part B: Clean Up of pcr product using Epoch BIoLabs GenCatch PCR CleanUp Kit==
		+	Before we can ligate our bacterial 16s rDNA into vector plasmids, we must remove interfering dNPTs, primers, and other small degraded DNA. We will use a column that separates DNA by size. Since the reagents and column materials in the kit we will use are proprietary, we won't know exactly what is going on at each step but, basicially, we will apply our pcr product to a column of a particular density, wash away elements too small to be trapped in it, and elute off the larger fragments of DNA (that should be ~1500bps if our pcr amplification of the 16s rRNA genes in our soil genomic DNA was successful).
	'''Notes before Starting:'''<BR>		'''Notes before Starting:'''<BR>
	95% ethanol has been added to Buffer WS before first time use (see bottle label for volume).<BR>		95% ethanol has been added to Buffer WS before first time use (see bottle label for volume).<BR>

Tucker Crum: /* Soil Bacterial Community Physiological Profiling (Culture Dependent Assessment) */

Tucker Crum — Wed, 16 Feb 2011 14:40:35 GMT

Soil Bacterial Community Physiological Profiling (Culture Dependent Assessment)

←Older revision		Revision as of 14:40, 16 February 2011
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	5. Calculate the % positive for the enzymatic activity for each assay (# positive colonies x dilution factor/total colony count x dilution factor [on nutrient agar] ) X 100. This correction for dilution factor allows you to compare the CFUs counted from different dilutions on plates. If you are able to use control (NA) and test plates from the same dilution (each has between 30-300 colonies), you can omit the dilution factor. This is the total number of CFUs/gram of wet soil of microorganisms able to perform the role of interest. <BR><BR>		5. Calculate the % positive for the enzymatic activity for each assay (# positive colonies x dilution factor/total colony count x dilution factor [on nutrient agar] ) X 100. This correction for dilution factor allows you to compare the CFUs counted from different dilutions on plates. If you are able to use control (NA) and test plates from the same dilution (each has between 30-300 colonies), you can omit the dilution factor. This is the total number of CFUs/gram of wet soil of microorganisms able to perform the role of interest. <BR><BR>
		+
		+	6. Add your data to the course spreadsheet on the instructor's computer. Be sure to click File Save after you enter your data.

	==PART E: Isolation of Azotobacter, Hyphomicrobia, Spore Forming, or other interesting Bacteria==		==PART E: Isolation of Azotobacter, Hyphomicrobia, Spore Forming, or other interesting Bacteria==

Tucker Crum: /* Part C: Agarose Gel Electrophoresis of Clean PCR PRODUCT */

Tucker Crum — Wed, 16 Feb 2011 14:37:19 GMT

Part C: Agarose Gel Electrophoresis of Clean PCR PRODUCT

←Older revision		Revision as of 14:37, 16 February 2011
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	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 TBE buffer (10x=890mM Tris, 890mM Boric Acid, 20mM EDTA) with SybrSafe™ stain.<BR><BR>	+	Your agarose gel is made of 1.0% agarose (w/v) in 1x TBE buffer (10x=890mM Tris, 890mM Boric Acid, 20mM 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>		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>

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

Tucker Crum — Wed, 16 Feb 2011 14:36:34 GMT

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

Janet McDonough: /* PART E: Isolation of Azotobacter, Hypomicrobia, Spore Forming, or other interesting Bacteria */

Janet McDonough — Tue, 15 Feb 2011 13:10:05 GMT

PART E: Isolation of Azotobacter, Hypomicrobia, Spore Forming, or other interesting Bacteria

←Older revision		Revision as of 13:10, 15 February 2011
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	5. Calculate the % positive for the enzymatic activity for each assay (# positive colonies x dilution factor/total colony count x dilution factor [on nutrient agar] ) X 100. This correction for dilution factor allows you to compare the CFUs counted from different dilutions on plates. If you are able to use control (NA) and test plates from the same dilution (each has between 30-300 colonies), you can omit the dilution factor. This is the total number of CFUs/gram of wet soil of microorganisms able to perform the role of interest. <BR><BR>		5. Calculate the % positive for the enzymatic activity for each assay (# positive colonies x dilution factor/total colony count x dilution factor [on nutrient agar] ) X 100. This correction for dilution factor allows you to compare the CFUs counted from different dilutions on plates. If you are able to use control (NA) and test plates from the same dilution (each has between 30-300 colonies), you can omit the dilution factor. This is the total number of CFUs/gram of wet soil of microorganisms able to perform the role of interest. <BR><BR>

-	==PART E: Isolation of Azotobacter, ~~Hypomicrobia~~, Spore Forming, or other interesting Bacteria==	+	==PART E: Isolation of Azotobacter, Hyphomicrobia, Spore Forming, or other interesting Bacteria==
	Continue to attempt to isolate to pure culture desired groups of bacteria. Directions found in the Protocols section of the wiki at [[BISC209/S11: Culture Media \| Cuture Media: General Purpose, Selective, Enrichment, Differential, & Assessment of Digestive Exo-Enzymes]]<BR>		Continue to attempt to isolate to pure culture desired groups of bacteria. Directions found in the Protocols section of the wiki at [[BISC209/S11: Culture Media \| Cuture Media: General Purpose, Selective, Enrichment, Differential, & Assessment of Digestive Exo-Enzymes]]<BR>
	Directions for Streaking for Isolation onto new solid media is found at [[BISC209/S11: Streaking for Isolation \| Streaking for Isolation ]]<BR>		Directions for Streaking for Isolation onto new solid media is found at [[BISC209/S11: Streaking for Isolation \| Streaking for Isolation ]]<BR>

Janet McDonough: /* Soil Bacterial Community Physiological Profiling (Culture Dependent Assessment) */

Janet McDonough — Tue, 15 Feb 2011 13:09:43 GMT

Soil Bacterial Community Physiological Profiling (Culture Dependent Assessment)

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	150/(0.1ml plated*1X10<sup>-3</sup>dilution)= 150X10<sup>4</sup> which in scientific notation is written as 1.5X10<sup>6</sup> CFU/gram <BR><BR>		150/(0.1ml plated*1X10<sup>-3</sup>dilution)= 150X10<sup>4</sup> which in scientific notation is written as 1.5X10<sup>6</sup> CFU/gram <BR><BR>

-	2. Flood the starch plate with a thin layer of iodine and count the number of colonies that show starch digestion activity as a clear zone or non-blue halo around the colony).<BR> <BR>	+	2. Flood the starch plate with a thin layer of Grams iodine and count the number of colonies that show starch digestion activity as a clear zone or non-blue halo around the colony).<BR> <BR>

	3. Count the number of colonies that show cellulose digestion activity as a clear zone or halo around the colony.<BR><BR>		3. Count the number of colonies that show cellulose digestion activity as a clear zone or halo around the colony.<BR><BR>

Janet McDonough: /* Part C: Agarose Gel Electrophoresis of Clean PCR PRODUCT */

Janet McDonough — Tue, 15 Feb 2011 13:08:25 GMT

Part C: Agarose Gel Electrophoresis of Clean PCR PRODUCT

←Older revision		Revision as of 13:08, 15 February 2011
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	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>	+	Your agarose gel is made of 1.0% agarose solution (w/v) in 1x TBE buffer (10x=890mM Tris, 890mM Boric Acid, 20mM 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>		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>
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	''' 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 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 at http://www.invitrogen.com). Record on the gel template in which well you have loaded your pcr product. 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>	+	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 1xTBE buffer with Sybr Safe DNA stain (a proprietary reagent from Invitrogen used according to manufacturer's directions at http://www.invitrogen.com). Record on the gel template in which well you have loaded your pcr product. 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>
-	Note that 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 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>	+	Note that 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 of agarose gel run time. 1x TBE buffer is used in this electrophoretic separation (89mM Tris, 89mM Boric acid, 2.0mM 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>		How will you judge a successful amplification? How many fragments and of what size do you expect to see? <BR><BR>

Janet McDonough: /* Part B: Clean Up of pcr product using Epoch BIoLabs GenCatch PCR CleanUp Kit */

Janet McDonough — Tue, 15 Feb 2011 12:44:12 GMT

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

Janet McDonough: /* Part A: PCR Amplification of 16s rRNA genes from Universal Bacterial Primers */

Janet McDonough — Tue, 15 Feb 2011 12:40:09 GMT

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

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	http://www.dnalc.org/resources/animations/pcr.html<BR><BR>		http://www.dnalc.org/resources/animations/pcr.html<BR><BR>

-	All PCR reactions require a thermal cycler to elevate and reduce the reaction temperature quickly and keep it at a specific temperature for a prescribed amount of time. There is a basic pattern to these temp. cycles, but there are differences, so you must be sure to program the cycler with the correct time and temperature for your specific amplification. Traditionally, pcr used Taq polymerase, a heat stable DNA polymerase originally found in a extremophilic bacterium, ''Thermus aquaticus'', that lives and reproduces in boiling hot springs. We are not using Taq for our pcr but a different polymerase, Finnzyme's Phusion High-Fidelity Polymerase, a proprietary reagent that uses a novel heat-stable ''Pyrococcus-like'' enzyme. Phusion DNA Polymerase generates long templates with an greater accuracy and speed than with Taq. The error rate of Phusion DNA Polymerase in Phusion HF Buffer is determined to be 4.4 x 10-7, which is approximately 50-fold lower than that of ''Thermus aquaticus'' DNA polymerase, and 6-fold lower than that of ''Pyrococcus furiosus'', another proof-reading DNA polymerase.	+	All PCR reactions require a thermal cycler to elevate and reduce the reaction temperature quickly and keep it at a specific temperature for a prescribed amount of time. There is a basic pattern to these temp. cycles, but there are differences, so you must be sure to program the cycler with the correct time and temperature for your specific amplification. Traditionally, pcr used Taq polymerase, a heat stable DNA polymerase originally found in a extremophilic bacterium, ''Thermus aquaticus'', that lives and reproduces in boiling hot springs. We are not using Taq for our pcr but a different polymerase, Finnzyme's Phusion High-Fidelity Polymerase, a proprietary reagent that uses a novel heat-stable ''Pyrococcus-like'' enzyme. Phusion DNA Polymerase generates long templates with a greater accuracy and speed than with Taq. The error rate of Phusion DNA Polymerase in Phusion HF Buffer is determined to be 4.4 x 10-7, which is approximately 50-fold lower than that of ''Thermus aquaticus'' DNA polymerase, and 6-fold lower than that of ''Pyrococcus furiosus'', another proof-reading DNA polymerase.
	Therefore, our pcr product DNA will have far fewer "mistakes" in the sequences that are replicated from template DNA. Our polymerase will also work much faster so our ~20 cycles will require less time than conventional Taq based pcr. <br><br>		Therefore, our pcr product DNA will have far fewer "mistakes" in the sequences that are replicated from template DNA. Our polymerase will also work much faster so our ~20 cycles will require less time than conventional Taq based pcr. <br><br>

←Older revision		Revision as of 14:36, 16 February 2011
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	1. Measure 500 μl of '''Buffer PX''' using your P1000 and add part of it to your pcr product and the rest to a clean microfuge tube. Using your P200 set to 200 μL, remove all the pcrProduct/buffer mix in the pcr tube and add it to the PX buffer in the microfuge tube. Close the cap of the microfuge tube and mix. <BR><BR>		1. Measure 500 μl of '''Buffer PX''' using your P1000 and add part of it to your pcr product and the rest to a clean microfuge tube. Using your P200 set to 200 μL, remove all the pcrProduct/buffer mix in the pcr tube and add it to the PX buffer in the microfuge tube. Close the cap of the microfuge tube and mix. <BR><BR>
	2. Place a GenCatch™ spin column in a provided 2 ml collection tube.<BR><BR>		2. Place a GenCatch™ spin column in a provided 2 ml collection tube.<BR><BR>
-	3. Load all of the pcr product/bufferPX mixture created in step 1 (up to a maximum of 700μL total volume) to the spin column and centrifuge for 60 sec. ~~THERE~~ <BR><BR>	+	3. Load all of the pcr product/bufferPX mixture created in step 1 (up to a maximum of 700μL total volume) to the spin column and centrifuge for 60 sec.<BR><BR>
-	4. Discard flow-through. Place the spin column back into the same collection tube.~~<BR><BR>~~	+	4. Discard flow-through. Place the spin column back into the same collection tube.
	(Collection tubes are re-used to reduce plastic waste.)<BR><BR>		(Collection tubes are re-used to reduce plastic waste.)<BR><BR>
-	5. If you had more than 700 μL volume of pcrProduct/bufferPX made in step 1, apply the remaining volume to the spin column and centrifuge for 1 minute. Discard the flow through and place the spin column back in the same collection tube. ~~If you applied all the pcr product to the spin column in step 3, skip this step and proceed to step 6.~~ <BR><BR>	+	5. If you applied all the pcr product to the spin column in step 3, skip this step and proceed to step 6. If you had more than 700 μL volume of pcrProduct/bufferPX made in step 1, apply the remaining volume to the spin column and centrifuge for 1 minute. Discard the flow-through and place the spin column back in the same collection tube. <BR><BR>
	6. Wash the spin column by adding 500 μL '''Buffer WF''' to the spin column and centrifuge for 60 sec. Be careful to use '''WF''' buffer!! <BR><BR>		6. Wash the spin column by adding 500 μL '''Buffer WF''' to the spin column and centrifuge for 60 sec. Be careful to use '''WF''' buffer!! <BR><BR>
	7. Discard flow-through and place the spin column back in the same collection tube.<BR><BR>		7. Discard flow-through and place the spin column back in the same collection tube.<BR><BR>
-	8. Wash the spin column by applying 700 μL of '''Buffer WS'''. '''Note that WS Buffer is different than the buffer used in step 6.''' Centrifuge the column for an additional 1 min. Check that the buffer flow through is ~~complete~~, re-spin if needed. Discard the flow through<BR><BR>	+	8. Wash the spin column by applying 700 μL of '''Buffer WS'''. '''Note that WS Buffer is different than the buffer used in step 6!!!''' Centrifuge the column for an additional 1 min. Check that ALL the buffer is in the flow-through, if there is buffer remaining in the spin-column, re-spin if needed. Discard the flow-through.<BR><BR>
-	9. Centrifuge the spin column in the same collection tube at full speed for 3 more minutes to remove ethanol residue. ''It is crucially important to remove all ethanol residue; residual ethanol may inhibit subsequent enzymatic reactions.''<BR><BR>	+	9. Centrifuge the spin-column in the same collection tube at full speed for 3 more minutes to remove ALL ethanol residue. ''It is crucially important to remove all ethanol residue; residual ethanol may inhibit subsequent enzymatic reactions.''<BR><BR>
	10. Place each spin column into a new, clean 1.5 ml microcentrifuge tube (not a collection tube).<BR><BR>		10. Place each spin column into a new, clean 1.5 ml microcentrifuge tube (not a collection tube).<BR><BR>
-	11. To elute DNA, add ~~50 μl~~ of the '''Elution Buffer EB''' (10 mM Tris·Cl, pH 8.5) to the center of each spin column membrane. Let it stand for 2 minutes to allow it completely adsorb and then centrifuge the spin column in the microfuge tube for 1 min at 17,900 x g (13,000 rpm). <BR><BR>	+	11. To elute DNA, add 50μl of the '''Elution Buffer EB''' (10 mM Tris·Cl, pH 8.5) to the center of each spin column membrane. Let it stand for 2 minutes to allow it completely adsorb and then centrifuge the spin column in the microfuge tube for 1 min at 17,900 x g (13,000 rpm). <BR><BR>
-	Keep your pcr product on ice until your instructor tells you that it's time to load the gel to determine the success of this amplification and clean-up. <BR><BR>	+	Keep your pcr product on ice until your instructor tells you that it's time to load the gel in order to determine the success of this amplification and clean-up. <BR><BR>
	''IMPORTANT NOTES for using this kit: Ensure that the elution buffer (EB) is dispensed directly onto the spin column membrane for complete elution of bound DNA. The average eluate volume is 48 μl from 50 μl elution buffer volume.''<BR>		''IMPORTANT NOTES for using this kit: Ensure that the elution buffer (EB) is dispensed directly onto the spin column membrane for complete elution of bound DNA. The average eluate volume is 48 μl from 50 μl elution buffer volume.''<BR>
	''Elution efficiency is dependent on pH. The maximum elution efficiency is achieved		''Elution efficiency is dependent on pH. The maximum elution efficiency is achieved

←Older revision		Revision as of 12:44, 15 February 2011
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	'''Procedure'''<BR>		'''Procedure'''<BR>
-	1. Measure 500 μl of '''Buffer PX''' using your P1000 and add part of it to your ~~thawed~~ pcr product and the rest to a clean microfuge tube. Using your P200 set to 200 μL, remove all the pcrProduct/buffer mix in the pcr tube and add it to the PX buffer in the microfuge tube. Close the cap of the microfuge tube and mix. <BR><BR>	+	1. Measure 500 μl of '''Buffer PX''' using your P1000 and add part of it to your pcr product and the rest to a clean microfuge tube. Using your P200 set to 200 μL, remove all the pcrProduct/buffer mix in the pcr tube and add it to the PX buffer in the microfuge tube. Close the cap of the microfuge tube and mix. <BR><BR>
	2. Place a GenCatch™ spin column in a provided 2 ml collection tube.<BR><BR>		2. Place a GenCatch™ spin column in a provided 2 ml collection tube.<BR><BR>
	3. Load all of the pcr product/bufferPX mixture created in step 1 (up to a maximum of 700μL total volume) to the spin column and centrifuge for 60 sec. THERE <BR><BR>		3. Load all of the pcr product/bufferPX mixture created in step 1 (up to a maximum of 700μL total volume) to the spin column and centrifuge for 60 sec. THERE <BR><BR>
	4. Discard flow-through. Place the spin column back into the same collection tube.<BR><BR>		4. Discard flow-through. Place the spin column back into the same collection tube.<BR><BR>
	(Collection tubes are re-used to reduce plastic waste.)<BR><BR>		(Collection tubes are re-used to reduce plastic waste.)<BR><BR>
-	5. If you had more than 700 μL volume of pcrProduct/bufferPX made in step 1, apply the remaining volume to the spin column and centrifuge for 1 minute. Discard the flow through and place the spin column back in the same collection tube. If you applied all the ~~pcrproduct~~ to the spin column in step 3, skip this step and proceed to step 6. <BR><BR>	+	5. If you had more than 700 μL volume of pcrProduct/bufferPX made in step 1, apply the remaining volume to the spin column and centrifuge for 1 minute. Discard the flow through and place the spin column back in the same collection tube. If you applied all the pcr product to the spin column in step 3, skip this step and proceed to step 6. <BR><BR>
	6. Wash the spin column by adding 500 μL '''Buffer WF''' to the spin column and centrifuge for 60 sec. Be careful to use '''WF''' buffer!! <BR><BR>		6. Wash the spin column by adding 500 μL '''Buffer WF''' to the spin column and centrifuge for 60 sec. Be careful to use '''WF''' buffer!! <BR><BR>
	7. Discard flow-through and place the spin column back in the same collection tube.<BR><BR>		7. Discard flow-through and place the spin column back in the same collection tube.<BR><BR>
-	8. Wash the column by applying 700 μL of '''Buffer WS''' ~~to the spin column~~. '''Note that WS Buffer is different than the buffer used in step 6.''' Centrifuge the column for an additional 1 min. Discard the flow through<BR><BR>	+	8. Wash the spin column by applying 700 μL of '''Buffer WS'''. '''Note that WS Buffer is different than the buffer used in step 6.''' Centrifuge the column for an additional 1 min. Check that the buffer flow through is complete, re-spin if needed. Discard the flow through<BR><BR>
	9. Centrifuge the spin column in the same collection tube at full speed for 3 more minutes to remove ethanol residue. ''It is crucially important to remove all ethanol residue; residual ethanol may inhibit subsequent enzymatic reactions.''<BR><BR>		9. Centrifuge the spin column in the same collection tube at full speed for 3 more minutes to remove ethanol residue. ''It is crucially important to remove all ethanol residue; residual ethanol may inhibit subsequent enzymatic reactions.''<BR><BR>
	10. Place each spin column into a new, clean 1.5 ml microcentrifuge tube (not a collection tube).<BR><BR>		10. Place each spin column into a new, clean 1.5 ml microcentrifuge tube (not a collection tube).<BR><BR>
	11. To elute DNA, add 50 μl of the '''Elution Buffer EB''' (10 mM Tris·Cl, pH 8.5) to the center of each spin column membrane. Let it stand for 2 minutes to allow it completely adsorb and then centrifuge the spin column in the microfuge tube for 1 min at 17,900 x g (13,000 rpm). <BR><BR>		11. To elute DNA, add 50 μl of the '''Elution Buffer EB''' (10 mM Tris·Cl, pH 8.5) to the center of each spin column membrane. Let it stand for 2 minutes to allow it completely adsorb and then centrifuge the spin column in the microfuge tube for 1 min at 17,900 x g (13,000 rpm). <BR><BR>
	Keep your pcr product on ice until your instructor tells you that it's time to load the gel to determine the success of this amplification and clean-up. <BR><BR>		Keep your pcr product on ice until your instructor tells you that it's time to load the gel to determine the success of this amplification and clean-up. <BR><BR>
-	''IMPORTANT NOTES for using this kit: Ensure that the elution buffer is dispensed directly onto the spin column membrane for complete elution of bound DNA. The average eluate volume is 48 μl from 50 μl elution buffer volume~~, and 28 μl from 30 μl elution buffer~~.''<BR>	+	''IMPORTANT NOTES for using this kit: Ensure that the elution buffer (EB) is dispensed directly onto the spin column membrane for complete elution of bound DNA. The average eluate volume is 48 μl from 50 μl elution buffer volume.''<BR>
	''Elution efficiency is dependent on pH. The maximum elution efficiency is achieved		''Elution efficiency is dependent on pH. The maximum elution efficiency is achieved
	between pH 7.0 and 8.5. Store DNA at –20°C as DNA may degrade in the absence of a buffering		between pH 7.0 and 8.5. Store DNA at –20°C as DNA may degrade in the absence of a buffering