BISC219/F12: RNAi Lab 8 - Revision history

Melissa Beers: /* Part 3: Transformation of isolated plasmid DNA into E. coli strain HT115(DE3) */

2012-11-01T17:51:09Z

Part 3: Transformation of isolated plasmid DNA into E. coli strain HT115(DE3)

←Older revision		Revision as of 17:51, 1 November 2012
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	#Add 5 microliters of the 50mg/ml ampicillin stock (also found in the refrigerator with the broth) to each tube. '''Calculate the effective concentration of ampicillin''' that you have in your LB tube (remember V1 x C1= V2 x C2) and record that information in your lab notebook. <br>		#Add 5 microliters of the 50mg/ml ampicillin stock (also found in the refrigerator with the broth) to each tube. '''Calculate the effective concentration of ampicillin''' that you have in your LB tube (remember V1 x C1= V2 x C2) and record that information in your lab notebook. <br>
	# Add 5 microliters of the 12.5mg/ml tetracycline stock (also found in the refrigerator with the broth). '''Calculate the effective concentration of tetracycline''' that you havein your LB broth tube. Record that info in your lab notebook.		# Add 5 microliters of the 12.5mg/ml tetracycline stock (also found in the refrigerator with the broth). '''Calculate the effective concentration of tetracycline''' that you havein your LB broth tube. Record that info in your lab notebook.
-	#Gently swirl your LB +amp broth to mix the contents. <br>	+	#Gently swirl your LB +amp+tet broth to mix the contents. <br>
	#Label the two sterile glass culture tubes with tape in your team color. Label one with "pPD129.36 ''lsy-2''" and your initials. Label the other with your initials only. <br>		#Label the two sterile glass culture tubes with tape in your team color. Label one with "pPD129.36 ''lsy-2''" and your initials. Label the other with your initials only. <br>
-	#Inoculate the broth with your bacteria by using a sterile disposable loop to scrape your candidate colony off the plate. Be sure not to touch the plate with the loop except on the desired colony and don’t pick up any satellite colonies! Gently swirl the loop in the LB+amp broth - you should be able to see the colony come off the loop. (The second tube of broth labeled with just your initials is a control and should not be inoculated with bacteria as it is your control for contamination.) If you prefer to use a sterile toothpick rather than a loop, you may pick up the colony with the sterile toothpick and drop the toothpick into the broth culture. Note that the tip with the colony is in the broth and the contaminated part you touched with your fingers does not touch the sterile medium.<br>	+	#Inoculate the broth with your bacteria by using a sterile disposable loop to scrape your candidate colony off the plate. Be sure not to touch the plate with the loop except on the desired colony and don’t pick up any satellite colonies! Gently swirl the loop in the LB+amp+tet broth - you should be able to see the colony come off the loop. (The second tube of broth labeled with just your initials is a control and should not be inoculated with bacteria as it is your control for contamination.) If you prefer to use a sterile toothpick rather than a loop, you may pick up the colony with the sterile toothpick and drop the toothpick into the broth culture. Note that the tip with the colony is in the broth and the contaminated part you touched with your fingers does not touch the sterile medium.<br>
	#Balance the 2 tubes across from each other on the rotating wheel in the 37C incubator at the front of the room when you come in the door. DO NOT USE THE ROOM TEMP WHEEL!		#Balance the 2 tubes across from each other on the rotating wheel in the 37C incubator at the front of the room when you come in the door. DO NOT USE THE ROOM TEMP WHEEL!
	#Incubate these broth cultures at 37°C overnight. '''Do not forget to make sure the wheel is rotating when you leave!'''<br>		#Incubate these broth cultures at 37°C overnight. '''Do not forget to make sure the wheel is rotating when you leave!'''<br>

Melissa Beers: /* Plasmid Isolation and Transformation */

2012-11-01T17:49:08Z

Plasmid Isolation and Transformation

←Older revision		Revision as of 17:49, 1 November 2012
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	Last week, you tested two colonies of previously transformed bacteria in order to find one that contains a plasmid with the ''C. elegans'' ''lsy-2'' gene. You have confirmed that the colony you have sub-cultured last night contains ''E. coli'' bacteria-- not just any pPD129.36 plasmid but a copy of pPD129.36+''lsy-2''. Now we can isolate the plasmid DNA from these cells and transform these plasmids into a different special strain of ''E. coli'' bacteria, HT115(DE3). We will use this strain because we can induce the HT115(DE3) strain to overexpress the plasmid genes, resulting in the presence of a lot of double stranded RNA specific to ''lsy-2,''our gene of interest in these bacteria.<br>		Last week, you tested two colonies of previously transformed bacteria in order to find one that contains a plasmid with the ''C. elegans'' ''lsy-2'' gene. You have confirmed that the colony you have sub-cultured last night contains ''E. coli'' bacteria-- not just any pPD129.36 plasmid but a copy of pPD129.36+''lsy-2''. Now we can isolate the plasmid DNA from these cells and transform these plasmids into a different special strain of ''E. coli'' bacteria, HT115(DE3). We will use this strain because we can induce the HT115(DE3) strain to overexpress the plasmid genes, resulting in the presence of a lot of double stranded RNA specific to ''lsy-2,''our gene of interest in these bacteria.<br>
	<br>		<br>
-	To recap: Yesterday you inoculated a few milliliters of LB broth containing ''E. coli'' that we are sure have maintained our genetically engineered plasmid, pPD129.36, modified to contain an antibiotic resistance gene to ampicillin and all or part of the ''C. elegans'' ''lsy-2''gene that you want to investigate. You added ampicillin to the broth to ensure that the plasmid DNA would be maintained by the cells. Overnight the bacteria have grown to high density and the plasmid DNA has undergone many replications. However since you started with a single colony of bacteria and that colony grew from a single transformed cell, all the copies of the plasmid DNA in your overnight culture should be identical (“clones” of one another). To isolate the plasmid DNA from this bacterial strain that expresses ''C. elegans lsy-2'' , you will perform what is commonly called a “mini-prep”. This term distinguishes the procedure from a “maxi-” or “large scale-prep” which involves a larger volume of cells and additional steps of purification. The overall goal of each “prep” is the same--to separate the plasmid DNA from the chromosomal DNA so that a certain gene on the plasmid DNA can be studied further.<br>	+	To recap: Yesterday you inoculated a few milliliters of LB broth containing ''E. coli'' that we are sure have maintained our genetically engineered plasmid, pPD129.36, modified to contain an antibiotic resistance gene to ampicillin and all or part of the ''C. elegans'' ''lsy-2'' gene that you want to investigate. You added ampicillin to the broth to ensure that the plasmid DNA would be maintained by the cells. Overnight the bacteria have grown to high density and the plasmid DNA has undergone many replications. However since you started with a single colony of bacteria and that colony grew from a single transformed cell, all the copies of the plasmid DNA in your overnight culture should be identical (“clones” of one another). To isolate the plasmid DNA from this bacterial strain that expresses ''C. elegans lsy-2'' , you will perform what is commonly called a “mini-prep”. This term distinguishes the procedure from a “maxi-” or “large scale-prep” which involves a larger volume of cells and additional steps of purification. The overall goal of each “prep” is the same--to separate the plasmid DNA from the chromosomal DNA so that a certain gene on the plasmid DNA can be studied further.<br>
	<br>		<br>
	'''TO DO TODAY:'''<br>		'''TO DO TODAY:'''<br>

Melissa Beers: /* Lab 8: Series 3- Creating the feeding strain of bacteria for RNAi in C. elegans */

2012-11-01T17:48:36Z

Lab 8: Series 3- Creating the feeding strain of bacteria for RNAi in C. elegans

←Older revision		Revision as of 17:48, 1 November 2012
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	#Begin by obtaining two tubes of LB broth (each will have 5 ml of broth) from the refrigerator in the back left hand corner of the room.<br>		#Begin by obtaining two tubes of LB broth (each will have 5 ml of broth) from the refrigerator in the back left hand corner of the room.<br>
	#Add 5 microliters of the 50mg/ml ampicillin stock (also found in the refrigerator) to each tube. You may use your P20 micropipet and the middle size tips or a P10 and the smallest tips. (Make sure that you adjust your pipet to the proper setting which will look different on a P10 vs. a P20!)'''Calculate the effective concentration of ampicillin''' that you will have in your LB tube (remember V1 x C1= V2 x C2) and record that information in your lab notebook. <br>		#Add 5 microliters of the 50mg/ml ampicillin stock (also found in the refrigerator) to each tube. You may use your P20 micropipet and the middle size tips or a P10 and the smallest tips. (Make sure that you adjust your pipet to the proper setting which will look different on a P10 vs. a P20!)'''Calculate the effective concentration of ampicillin''' that you will have in your LB tube (remember V1 x C1= V2 x C2) and record that information in your lab notebook. <br>
-	#Add 5 microliters of the 12.5mg/ml tetracycline stock(also found in the refrigerator) to each tube. You may use your P20 micropipet and the middle size tips or a P10 and the smallest tips. '''Calculate the effective concentration of tetracycline''' that you will have in your LB tube (remember V1 x C1= V2 x C2) and record that information in your lab notebook. <br>	+	#Gently swirl your LB +amp broth to mix the contents. DO NOT invert the culture tubes as the tops are not spill resistant! <br>
-	#Gently swirl your LB +amp ~~+ tet~~ broth to mix the contents. DO NOT invert the culture tubes as the tops are not spill resistant! <br>	+
	#Label the two sterile glass culture tubes with tape in your team color. Label one with "pPD129.36 ''lsy-2''" and your initials. Label the other with your initials only. <br>		#Label the two sterile glass culture tubes with tape in your team color. Label one with "pPD129.36 ''lsy-2''" and your initials. Label the other with your initials only. <br>
-	#Inoculate the broth with your bacteria by using a sterile disposable loop or a sterile toothpick (both found in the supply area near the door to L304) to scrape your candidate colony off the plate. Be sure not to touch the plate with the loop except on the desired colony. Try to avoid picking up any satellite colonies or another colony nearby! Gently swirl the loop in the LB+amp~~+tet~~ broth - you should be able to see the colony come off the loop. If you use a sterile toothpick, you can just drop it into the broth so that the tip with the colony is in the broth. Note thatthe part you touched does not come in contact with the medium. (The second tube of broth labeled with just your initials is a control and should not be inoculated with bacteria as it is your control for contamination.) <br>	+	#Inoculate the broth with your bacteria by using a sterile disposable loop or a sterile toothpick (both found in the supply area near the door to L304) to scrape your candidate colony off the plate. Be sure not to touch the plate with the loop except on the desired colony. Try to avoid picking up any satellite colonies or another colony nearby! Gently swirl the loop in the LB+amp broth - you should be able to see the colony come off the loop. If you use a sterile toothpick, you can just drop it into the broth so that the tip with the colony is in the broth. Note thatthe part you touched does not come in contact with the medium. (The second tube of broth labeled with just your initials is a control and should not be inoculated with bacteria as it is your control for contamination.) <br>
	#Balance the 2 tubes across from each other on the rotating wheel in the 37C incubator at the front of the room when you come in the door. DO NOT USE THE wheel that is at Room TEMP!		#Balance the 2 tubes across from each other on the rotating wheel in the 37C incubator at the front of the room when you come in the door. DO NOT USE THE wheel that is at Room TEMP!
	#Incubate these broth cultures at 37°C overnight. '''Do not forget to make sure the wheel is rotating when you leave!'''<br>		#Incubate these broth cultures at 37°C overnight. '''Do not forget to make sure the wheel is rotating when you leave!'''<br>

Tucker Crum: /* Part 3: Transformation of isolated plasmid DNA into E. coli strain HT115(DE3) */

2012-11-01T12:08:58Z

Part 3: Transformation of isolated plasmid DNA into E. coli strain HT115(DE3)

←Older revision		Revision as of 12:08, 1 November 2012
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	#Obtain a tube containing 50 μL of competent cells from your instructor.<br>		#Obtain a tube containing 50 μL of competent cells from your instructor.<br>
	#Label the top or the side of the tube with HT115(DE3), pPD12936+''lsy-2'', and your initials or team color. <br>		#Label the top or the side of the tube with HT115(DE3), pPD12936+''lsy-2'', and your initials or team color. <br>
-	#~~Start~~ the transformation by '''~~pipetting~~ 10μL ~~(250 nanograms)~~ of your plasmid DNA''' to the tube. Pipet up and down once to mix the DNA and the cells. Close the cap and let the transformation mixture sit '''on ice for 10 minutes'''. <br>	+	#Consult with your instructor to determine how much of your plasmid DNA you should add to the cells to start the transformation. When you have determined the correct vol., '''pipet between 1μL and 10μL of your plasmid DNA''' to the tube. (The volume you should use is dependent on the conc. you achieved in your mini-prep.) Pipet up and down once to mix the DNA and the cells. Close the cap and let the transformation mixture sit '''on ice for 10 minutes'''. <br>
	#Heat shock by incubating the transformation mix at '''42°C for 90 seconds''', exactly. This step must be timed exactly. Remove the tube at the end of 90 seconds to your ice bucket while you get your LB ready. <br>		#Heat shock by incubating the transformation mix at '''42°C for 90 seconds''', exactly. This step must be timed exactly. Remove the tube at the end of 90 seconds to your ice bucket while you get your LB ready. <br>
-	#~~Add~~ '''250 microliters ~~of LB~~''' ~~in your conical tube~~ to the transformation mix. LB aliquots should be in the 37°C incubator. When pipetting the media, remember to release your thumb on your micropipet slowly, to avoid splashing the liquid on the end of the barrel. The barrel is not sterile and if you see the liquid touch it, then discard the media in the waste beaker and try again with a new tip. <br>	+	#Consult your instructor to determine the volume of '''warm LB broth you should add (usually between 250-500 microliters)''' to the transformation mix. LB aliquots should be found in the 37°C mixer/incubator on the green/blue team bench. When pipetting the media, remember to release your thumb on your micropipet slowly, to avoid splashing the liquid on the end of the barrel. The barrel is not sterile and if you see the liquid touch it, then discard the media in the waste beaker and try again with a new tip. <br>
-	#Once you have added the LB, close the cap and invert the tube once or twice to mix the contents. '''Incubate at 37°C for 30-45 minutes ~~with very gentle mixing~~ in the bench top incubator mixer.''' <br>	+	#Once you have added the LB, close the cap and invert the tube once or twice to mix the contents. '''Incubate at 37°C for 30-45 minutes in the bench top incubator/ mixer with occasional gentle mixing.''' <br>
	#While the plasmid DNA is being taken up by the competent cells and the new genes provided by the plasmid are being expressed by the bacteria, label two LB + amp agar plates. Label the bottom of the plated with the strain's identity (HT115(DE3)), the plasmid used, the date, your initials and team color. You must label the bottom of the plated since the tops are easily switched. Differentiate them by putting 50μL on one and 200μL on the other. Put these plates in the hood with the blower on and with the lid ajar to dry the surface of the agar for about 10 minutes or until the surface looks dry but is not badly dehydrated.<br>		#While the plasmid DNA is being taken up by the competent cells and the new genes provided by the plasmid are being expressed by the bacteria, label two LB + amp agar plates. Label the bottom of the plated with the strain's identity (HT115(DE3)), the plasmid used, the date, your initials and team color. You must label the bottom of the plated since the tops are easily switched. Differentiate them by putting 50μL on one and 200μL on the other. Put these plates in the hood with the blower on and with the lid ajar to dry the surface of the agar for about 10 minutes or until the surface looks dry but is not badly dehydrated.<br>
	#Once the transformation mix has incubated at '''37°C for 30-45 minutes''', invert it to mix the contents and '''pipet 50 microliters of transformed cells''' onto the center of a labeled slightly dehydrated LB + amp plate prepared in the previous step. '''Pipet 200μL on the other plate'''. Pour 5-10 glass beads onto the plates. Put the lid back on and gently swirl the beads all over the plates to spread the transformed bacteria around. When you are done - pour the beads into the beaker with disinfectant near the sink.		#Once the transformation mix has incubated at '''37°C for 30-45 minutes''', invert it to mix the contents and '''pipet 50 microliters of transformed cells''' onto the center of a labeled slightly dehydrated LB + amp plate prepared in the previous step. '''Pipet 200μL on the other plate'''. Pour 5-10 glass beads onto the plates. Put the lid back on and gently swirl the beads all over the plates to spread the transformed bacteria around. When you are done - pour the beads into the beaker with disinfectant near the sink.

Tucker Crum: /* Measuring the concentration of plasmid DNA using the NanoDropper */

2012-11-01T12:02:54Z

Measuring the concentration of plasmid DNA using the NanoDropper

←Older revision		Revision as of 12:02, 1 November 2012
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	''Note that as in traditional spectroscopy, the blank will be subtracted from subsequent measurements. If you want to determine the contribution of a specific buffer or diluent, measure the buffer first using distilled water as a blank. If the buffer does not contribute to the A 260nm reading, then deionized water is fine to use as the blank. The water or buffer should always be measured to be sure that the instrument has been zeroed properly. The measurement of water or buffer should be zero or very close. All measurements are automatically normalized to 340nm.''<BR><BR>		''Note that as in traditional spectroscopy, the blank will be subtracted from subsequent measurements. If you want to determine the contribution of a specific buffer or diluent, measure the buffer first using distilled water as a blank. If the buffer does not contribute to the A 260nm reading, then deionized water is fine to use as the blank. The water or buffer should always be measured to be sure that the instrument has been zeroed properly. The measurement of water or buffer should be zero or very close. All measurements are automatically normalized to 340nm.''<BR><BR>

-	5. Measure the nucleic acid sample by loading 1microliter of sample and selecting "measure". Record your DNA concentration. Once the measurement is complete. Clean both optical surfaces with a Kimwipe and the machine is ready for the next sample. <br>	+	5. Measure the nucleic acid sample by loading 1microliter of sample and selecting "measure". Record your DNA concentration in your lab notebook and on the tube of plamid DNA. Once the measurement is complete. Clean both optical surfaces with a Kimwipe and the machine is ready for the next sample. <br>
	''You should ensure that the appropriate constant (50 for dsDNA or 40 for RNA) has been chosen. The software automatically calculates the nucleic acid concentration. If the calculation is done by hand, the A260nm is represented as a 1cm path for convenience, even though 1-nm and 0.2nm paths are actually used during the measurement cycle.''<BR><BR>		''You should ensure that the appropriate constant (50 for dsDNA or 40 for RNA) has been chosen. The software automatically calculates the nucleic acid concentration. If the calculation is done by hand, the A260nm is represented as a 1cm path for convenience, even though 1-nm and 0.2nm paths are actually used during the measurement cycle.''<BR><BR>

	'''Clean Up'''		'''Clean Up'''
	When the last sample was been measured, clean the sampling device by repeating step 1. <BR><BR>		When the last sample was been measured, clean the sampling device by repeating step 1. <BR><BR>
-
-

	=='''Part 3: Transformation of isolated plasmid DNA into ''E. coli'' strain HT115(DE3)'''==		=='''Part 3: Transformation of isolated plasmid DNA into ''E. coli'' strain HT115(DE3)'''==

Tucker Crum: /* Diluting your plasmid DNA */

2012-11-01T12:01:43Z

Diluting your plasmid DNA

←Older revision		Revision as of 12:01, 1 November 2012
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	When the last sample was been measured, clean the sampling device by repeating step 1. <BR><BR>		When the last sample was been measured, clean the sampling device by repeating step 1. <BR><BR>

-	~~=='''Diluting your plasmid DNA'''==~~	+
-	We hope that your mini-prep yielded a concentration of DNA much greater than 250ng/μL because we will need to use 250ng of DNA in each of our transformations that you will perform next. Calculate how to dilute your plasmid DNA in ultrapurified water to a total volume of 100μL (or 50μL if your conc. is low or more than 100μL if your conc. is very high) so that you can add 10μL of plasmid DNA to each of the transformations described below and achieve the desired addition of 250nanograms of DNA. '''Show your instructor your calculations BEFORE proceeding with the transformation!'''Hint: Remember your old friend, the formula V1 x C1 = V2 x C2. In this case V1 = your unknown vol of your stock DNA to add to achieve the total vol. desired; C1= the concentration the nanodropper gave you; V2= whatever total vol. you think is most appropriate for making this dilution (you want to be able to measure V1 accurately with a micropipet and you can't add more vol. than you have); C2= 25ng/μL (since you want to add 10μL of your plasmid DNA to achieve a total of 250ng of DNA). <BR><BR>	+

	=='''Part 3: Transformation of isolated plasmid DNA into ''E. coli'' strain HT115(DE3)'''==		=='''Part 3: Transformation of isolated plasmid DNA into ''E. coli'' strain HT115(DE3)'''==

Tucker Crum: /* Part 3: Transformation of isolated plasmid DNA into E. coli strain HT115(DE3) */

2012-10-31T13:20:03Z

Part 3: Transformation of isolated plasmid DNA into E. coli strain HT115(DE3)

←Older revision		Revision as of 13:20, 31 October 2012
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	#Obtain a tube containing 50 μL of competent cells from your instructor.<br>		#Obtain a tube containing 50 μL of competent cells from your instructor.<br>
	#Label the top or the side of the tube with HT115(DE3), pPD12936+''lsy-2'', and your initials or team color. <br>		#Label the top or the side of the tube with HT115(DE3), pPD12936+''lsy-2'', and your initials or team color. <br>
-	#Start the transformation by ~~pipetting~~ '''250 nanograms of your plasmid DNA''' to the tube. Pipet up and down once to mix the DNA and the cells. Close the cap and let the transformation mixture sit '''on ice for 10 minutes'''. <br>	+	#Start the transformation by '''pipetting 10μL (250 nanograms) of your plasmid DNA''' to the tube. Pipet up and down once to mix the DNA and the cells. Close the cap and let the transformation mixture sit '''on ice for 10 minutes'''. <br>
	#Heat shock by incubating the transformation mix at '''42°C for 90 seconds''', exactly. This step must be timed exactly. Remove the tube at the end of 90 seconds to your ice bucket while you get your LB ready. <br>		#Heat shock by incubating the transformation mix at '''42°C for 90 seconds''', exactly. This step must be timed exactly. Remove the tube at the end of 90 seconds to your ice bucket while you get your LB ready. <br>
-	#Add '''~~500~~ microliters of LB''' in your conical tube to the transformation mix. LB aliquots should be in the 37°C incubator. When pipetting the media, remember to release your thumb on your micropipet slowly, to avoid splashing the liquid on the end of the barrel. The barrel is not sterile and if you see the liquid touch it, then discard the media in the waste beaker and try again with a new tip. <br>	+	#Add '''250 microliters of LB''' in your conical tube to the transformation mix. LB aliquots should be in the 37°C incubator. When pipetting the media, remember to release your thumb on your micropipet slowly, to avoid splashing the liquid on the end of the barrel. The barrel is not sterile and if you see the liquid touch it, then discard the media in the waste beaker and try again with a new tip. <br>
	#Once you have added the LB, close the cap and invert the tube once or twice to mix the contents. '''Incubate at 37°C for 30-45 minutes with very gentle mixing in the bench top incubator mixer.''' <br>		#Once you have added the LB, close the cap and invert the tube once or twice to mix the contents. '''Incubate at 37°C for 30-45 minutes with very gentle mixing in the bench top incubator mixer.''' <br>
	#While the plasmid DNA is being taken up by the competent cells and the new genes provided by the plasmid are being expressed by the bacteria, label two LB + amp agar plates. Label the bottom of the plated with the strain's identity (HT115(DE3)), the plasmid used, the date, your initials and team color. You must label the bottom of the plated since the tops are easily switched. Differentiate them by putting 50μL on one and 200μL on the other. Put these plates in the hood with the blower on and with the lid ajar to dry the surface of the agar for about 10 minutes or until the surface looks dry but is not badly dehydrated.<br>		#While the plasmid DNA is being taken up by the competent cells and the new genes provided by the plasmid are being expressed by the bacteria, label two LB + amp agar plates. Label the bottom of the plated with the strain's identity (HT115(DE3)), the plasmid used, the date, your initials and team color. You must label the bottom of the plated since the tops are easily switched. Differentiate them by putting 50μL on one and 200μL on the other. Put these plates in the hood with the blower on and with the lid ajar to dry the surface of the agar for about 10 minutes or until the surface looks dry but is not badly dehydrated.<br>

Tucker Crum: /* Measuring the concentration of plasmid DNA using the NanoDropper */

2012-10-31T13:18:35Z

Measuring the concentration of plasmid DNA using the NanoDropper

←Older revision		Revision as of 13:18, 31 October 2012
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	3. Initialize the machine by placing 1 microliter of clean deionized water onto the lower optic surface, lower the lever arm, and select ''initialize'' from the NanoDrop software. Once initialization is complete (~10sec.), clean both optical surfaces with a Kimwipe. <BR><BR>		3. Initialize the machine by placing 1 microliter of clean deionized water onto the lower optic surface, lower the lever arm, and select ''initialize'' from the NanoDrop software. Once initialization is complete (~10sec.), clean both optical surfaces with a Kimwipe. <BR><BR>
	[[Image:sampleapply.jpg]]<BR>		[[Image:sampleapply.jpg]]<BR>
-	4. Perform a blank measurement by loading 1 microliter of ~~Solution 6 (10mM Tris) and select Blank. Note that this blanking step may use something other than Tris depending on what your sample is dissolve in. Often the blank will be~~ deionized ~~water if you have concentrated your DNA sample already with the ethanol precipation and resolubilized it in~~ water. <BR>	+	4. Perform a blank measurement by loading 1 microliter of purified deionized water. <BR>
-	''Note that as in traditional spectroscopy, the blank will be subtracted from subsequent measurements. If you want to determine the contribution of a specific buffer or diluent, measure the buffer first using distilled water as a blank. If the buffer does not contribute to the A 260nm reading, then deionized water ~~will be~~ fine to use as the blank. The water or buffer should always be measured to be sure that the instrument has been zeroed properly. The measurement of water or buffer should be zero or very close. All measurements are automatically normalized to 340nm.''<BR><BR>	+	''Note that as in traditional spectroscopy, the blank will be subtracted from subsequent measurements. If you want to determine the contribution of a specific buffer or diluent, measure the buffer first using distilled water as a blank. If the buffer does not contribute to the A 260nm reading, then deionized water is fine to use as the blank. The water or buffer should always be measured to be sure that the instrument has been zeroed properly. The measurement of water or buffer should be zero or very close. All measurements are automatically normalized to 340nm.''<BR><BR>

	5. Measure the nucleic acid sample by loading 1microliter of sample and selecting "measure". Record your DNA concentration. Once the measurement is complete. Clean both optical surfaces with a Kimwipe and the machine is ready for the next sample. <br>		5. Measure the nucleic acid sample by loading 1microliter of sample and selecting "measure". Record your DNA concentration. Once the measurement is complete. Clean both optical surfaces with a Kimwipe and the machine is ready for the next sample. <br>

Tucker Crum: /* Part 1: Plasmid DNA Isolation */

2012-10-31T13:17:24Z

Part 1: Plasmid DNA Isolation

←Older revision		Revision as of 13:17, 31 October 2012
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	#Check to be sure that your cells have not settled. If they have, mix the cultures but do NOT invert them because they will leak. Then pour some of the overnight culture so that both microfuge tubes are almost full. If you are nervous about pouring the bacteria, you can pipet 750 microliters into each tube '''twice''', so that there is a total of 1.5 ml in each tube. The exact volume doesn’t matter but the tubes should be quite full when you close the cap. <br>		#Check to be sure that your cells have not settled. If they have, mix the cultures but do NOT invert them because they will leak. Then pour some of the overnight culture so that both microfuge tubes are almost full. If you are nervous about pouring the bacteria, you can pipet 750 microliters into each tube '''twice''', so that there is a total of 1.5 ml in each tube. The exact volume doesn’t matter but the tubes should be quite full when you close the cap. <br>
	#Place the tubes in the room temperature microfuge so that the hinges of each cap is facing out. Paying attention to this small detail will help you know where in the tube to find your pellets. While it is not essential to do this in this step, it is a good habit to get into since sometimes pellets are very small and hard to see. Be sure your tubes are balanced, then '''spin the tubes for two minutes at 8,000 rpm'''. Check the rcf speed of the centrifuge while it is spinning (by hitting the toggle that changes the readout) and record the rcf’s in your lab notebook. Relative centrifugal force (rcf) is what should be used in M&M to describe your speed since it is universal and rpm is rotor and centrifuge dependent. <br>		#Place the tubes in the room temperature microfuge so that the hinges of each cap is facing out. Paying attention to this small detail will help you know where in the tube to find your pellets. While it is not essential to do this in this step, it is a good habit to get into since sometimes pellets are very small and hard to see. Be sure your tubes are balanced, then '''spin the tubes for two minutes at 8,000 rpm'''. Check the rcf speed of the centrifuge while it is spinning (by hitting the toggle that changes the readout) and record the rcf’s in your lab notebook. Relative centrifugal force (rcf) is what should be used in M&M to describe your speed since it is universal and rpm is rotor and centrifuge dependent. <br>
-	#If the supernatant is clear, pour it into the waste beaker that is on your bench, then flick the tube with the cap open to remove the last few drops of liquid. The cell pellet will not fall out. ~~If the supernatant is not clear, recentrifuge until it is.~~ <br>	+	#If the supernatant is not clear, recentrifuge until it is. When the supernatant is clear, pour it into the waste beaker that is on your bench, then flick the tube with the cap open to remove the last few drops of liquid. The cell pellet will not fall out. <br>
	#Resuspend your cell pellets COMPLETELY in '''100 microliters of solution I'''. Pipet up and down until ALL of the cells are uniformly suspended in Solution I. Be sure to change tips between samples. Leave the cells at room temperature as you prepare solution II.<br>		#Resuspend your cell pellets COMPLETELY in '''100 microliters of solution I'''. Pipet up and down until ALL of the cells are uniformly suspended in Solution I. Be sure to change tips between samples. Leave the cells at room temperature as you prepare solution II.<br>
	#To make solution II, '''mix 500 microliters of 2% SDS with 500 microliters of 0.4M NaOH in a microfuge tube'''. Close the cap and invert the tube several times to mix the contents. Add 200 microliters of solution II to each miniprep and invert the tubes five or six times to mix. In some cases the minipreps may appear to "clear" but don't worry if you don't see a big change in yours. Place the tubes on ice for '''five minutes'''.<br>		#To make solution II, '''mix 500 microliters of 2% SDS with 500 microliters of 0.4M NaOH in a microfuge tube'''. Close the cap and invert the tube several times to mix the contents. Add 200 microliters of solution II to each miniprep and invert the tubes five or six times to mix. In some cases the minipreps may appear to "clear" but don't worry if you don't see a big change in yours. Place the tubes on ice for '''five minutes'''.<br>

Tucker Crum: /* Plasmid Isolation and Transformation */

2012-10-31T13:16:44Z

Plasmid Isolation and Transformation

←Older revision		Revision as of 13:16, 31 October 2012
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	== Plasmid Isolation and Transformation ==		== Plasmid Isolation and Transformation ==
-	Last week, you tested two colonies of previously transformed bacteria in order to find one that contains a plasmid with the ''C. elegans'' ''lsy-2'' gene. ~~Now that you~~ have confirmed that the colony you have sub-cultured last night contains ''E. coli'' bacteria ~~that contains,~~ not just any pPD129.36 plasmid but a copy of pPD129.36+''lsy-2''. Now we can isolate the plasmid DNA from these cells and transform these plasmids into a different special strain of ''E. coli'' bacteria, HT115(DE3). We will use this strain because we can induce the HT115(DE3) strain to overexpress the plasmid genes, resulting in the presence of a lot of double stranded RNA specific to ''lsy-2,''our gene of interest in these bacteria.<br>	+	Last week, you tested two colonies of previously transformed bacteria in order to find one that contains a plasmid with the ''C. elegans'' ''lsy-2'' gene. You have confirmed that the colony you have sub-cultured last night contains ''E. coli'' bacteria-- not just any pPD129.36 plasmid but a copy of pPD129.36+''lsy-2''. Now we can isolate the plasmid DNA from these cells and transform these plasmids into a different special strain of ''E. coli'' bacteria, HT115(DE3). We will use this strain because we can induce the HT115(DE3) strain to overexpress the plasmid genes, resulting in the presence of a lot of double stranded RNA specific to ''lsy-2,''our gene of interest in these bacteria.<br>
	<br>		<br>
	To recap: Yesterday you inoculated a few milliliters of LB broth containing ''E. coli'' that we are sure have maintained our genetically engineered plasmid, pPD129.36, modified to contain an antibiotic resistance gene to ampicillin and all or part of the ''C. elegans'' ''lsy-2''gene that you want to investigate. You added ampicillin to the broth to ensure that the plasmid DNA would be maintained by the cells. Overnight the bacteria have grown to high density and the plasmid DNA has undergone many replications. However since you started with a single colony of bacteria and that colony grew from a single transformed cell, all the copies of the plasmid DNA in your overnight culture should be identical (“clones” of one another). To isolate the plasmid DNA from this bacterial strain that expresses ''C. elegans lsy-2'' , you will perform what is commonly called a “mini-prep”. This term distinguishes the procedure from a “maxi-” or “large scale-prep” which involves a larger volume of cells and additional steps of purification. The overall goal of each “prep” is the same--to separate the plasmid DNA from the chromosomal DNA so that a certain gene on the plasmid DNA can be studied further.<br>		To recap: Yesterday you inoculated a few milliliters of LB broth containing ''E. coli'' that we are sure have maintained our genetically engineered plasmid, pPD129.36, modified to contain an antibiotic resistance gene to ampicillin and all or part of the ''C. elegans'' ''lsy-2''gene that you want to investigate. You added ampicillin to the broth to ensure that the plasmid DNA would be maintained by the cells. Overnight the bacteria have grown to high density and the plasmid DNA has undergone many replications. However since you started with a single colony of bacteria and that colony grew from a single transformed cell, all the copies of the plasmid DNA in your overnight culture should be identical (“clones” of one another). To isolate the plasmid DNA from this bacterial strain that expresses ''C. elegans lsy-2'' , you will perform what is commonly called a “mini-prep”. This term distinguishes the procedure from a “maxi-” or “large scale-prep” which involves a larger volume of cells and additional steps of purification. The overall goal of each “prep” is the same--to separate the plasmid DNA from the chromosomal DNA so that a certain gene on the plasmid DNA can be studied further.<br>