20.109(S13):Induce protein and evaluate DNA (Day5) - Revision history

AgiStachowiak: /* Part 2: Run diagnostic gel */

AgiStachowiak — Mon, 01 Apr 2013 15:37:40 GMT

Part 2: Run diagnostic gel

←Older revision		Revision as of 15:37, 1 April 2013
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	===Part 2: Run diagnostic gel===		===Part 2: Run diagnostic gel===

-	The scheme below assumes that both Digest 1 (D1, used to analyze the positive control mutant) and Digest 2 (D2, used to analyze your X#Z mutant candidates) use only one enzyme. If you are doing double-digests and need to run single enzyme controls, hopefully you spoke to the teaching faculty about this plan last time. Load your samples on a 1% agarose gel in the following order, using 10 μL per ladder and 20 μL per plasmid:	+	The scheme below assumes that both Digest 1 (D1, used to analyze the positive control mutant) and Digest 2 (D2, used to analyze your X#Z mutant candidates) use only one enzyme. If you are doing double-digests and need to run single enzyme controls, hopefully you spoke to the teaching faculty about this plan last time. Load your samples on a 1% agarose gel (or a higher-weight percent gel, for those groups who are expecting a small band size) in the following order, using 10 μL per ladder and 20 μL per plasmid:

	<center>		<center>

AgiStachowiak: /* Part 3: Analyze sequence data */

AgiStachowiak — Sat, 30 Mar 2013 17:25:28 GMT

Part 3: Analyze sequence data

←Older revision		Revision as of 17:25, 30 March 2013
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	Your goal today is to analyze the sequencing data for three samples - two independent colonies from your X#Z mutant, and one reference mutant clone for practice - and then decide which colony to proceed with for the X#Z mutant.		Your goal today is to analyze the sequencing data for three samples - two independent colonies from your X#Z mutant, and one reference mutant clone for practice - and then decide which colony to proceed with for the X#Z mutant.

-	~~<font color=red>Update sequencing info</font color>~~
	You will want to have the [[Media: S12-M2-20109_pRSET-IPC.gb\| pRSET-IPC ApE file]] handy, and to mark and/or note down the expected location of your mutation before proceeding. (Just compare to your annotation of the IPC alone ApE file that you prepared on Day 1 of the module.) The data from Genewiz is available at [http://genewiz.com this link]. Choose the "Login" link and then use "astachow@mit.edu" and "be20109" to log in. At the bottom right should be a link to download your sequencing results.		You will want to have the [[Media: S12-M2-20109_pRSET-IPC.gb\| pRSET-IPC ApE file]] handy, and to mark and/or note down the expected location of your mutation before proceeding. (Just compare to your annotation of the IPC alone ApE file that you prepared on Day 1 of the module.) The data from Genewiz is available at [http://genewiz.com this link]. Choose the "Login" link and then use "astachow@mit.edu" and "be20109" to log in. At the bottom right should be a link to download your sequencing results.

AgiStachowiak: /* Part 3: Analyze sequence data */

AgiStachowiak — Sat, 30 Mar 2013 17:25:01 GMT

Part 3: Analyze sequence data

←Older revision		Revision as of 17:25, 30 March 2013
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	You will want to have the [[Media: S12-M2-20109_pRSET-IPC.gb\| pRSET-IPC ApE file]] handy, and to mark and/or note down the expected location of your mutation before proceeding. (Just compare to your annotation of the IPC alone ApE file that you prepared on Day 1 of the module.) The data from Genewiz is available at [http://genewiz.com this link]. Choose the "Login" link and then use "astachow@mit.edu" and "be20109" to log in. At the bottom right should be a link to download your sequencing results.		You will want to have the [[Media: S12-M2-20109_pRSET-IPC.gb\| pRSET-IPC ApE file]] handy, and to mark and/or note down the expected location of your mutation before proceeding. (Just compare to your annotation of the IPC alone ApE file that you prepared on Day 1 of the module.) The data from Genewiz is available at [http://genewiz.com this link]. Choose the "Login" link and then use "astachow@mit.edu" and "be20109" to log in. At the bottom right should be a link to download your sequencing results.

-	~~Select order~~ date 04-04-~~2012 for~~ T/R ~~results~~ and 04-05-~~2012 for~~ W/F ~~results~~. ~~You will all begin with~~ the ~~T/R link, which includes~~ data ~~for the three positive controls; start by analyzing the one you are working with, then move on to your unique mutant~~. The quickest way to start working with your data is to follow the "View" link under the Seq File heading. For ambiguous data, you may want to look directly at the Trace File as well.	+	Begin by analyzing your reference mutant, and then move on to your unique mutant.
		+	* Order date 03-25-13, #10-220995825 has T/R data as well as some reference mutant data. In particular, sample 3 is D24H and sample 4 is E67K (''not'' WT and D24H as written).
		+	* Order date 03-25-13, #10-220996747 has W/F data.
		+	* Order date 04-04-2012 (select "More..." and then use the search function) has T79P and M124S data.
		+	The quickest way to start working with your data is to follow the "View" link under the Seq File heading. For ambiguous data, you may want to look directly at the Trace File as well.

	Recall from Module 1 that you can align your sequencing data with a known sequence, in this case inverse pericam, and the differences will be quickly identified. There are several web-based programs for aligning sequences and still more programs that can be purchased. The steps for using one web-based tool, the same one you used in Module 1, are sketched below. Another popular alignment program is [http://www.clustal.org CLUSTAL].		Recall from Module 1 that you can align your sequencing data with a known sequence, in this case inverse pericam, and the differences will be quickly identified. There are several web-based programs for aligning sequences and still more programs that can be purchased. The steps for using one web-based tool, the same one you used in Module 1, are sketched below. Another popular alignment program is [http://www.clustal.org CLUSTAL].

AgiStachowiak: /* Reagent List */

AgiStachowiak — Thu, 28 Mar 2013 17:54:26 GMT

Reagent List

←Older revision		Revision as of 17:54, 28 March 2013
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	**RNase		**RNase

-	*1% and 1.2% agarose gels with ~~0.3 μg/mL ethidium bromide~~	+	*1% and 1.2% agarose gels with SYBR Safe

	*Gels made and run in 1X TAE buffer		*Gels made and run in 1X TAE buffer

AgiStachowiak: /* Part 6: Preparation for next time */

AgiStachowiak — Thu, 28 Mar 2013 17:46:40 GMT

Part 6: Preparation for next time

←Older revision		Revision as of 17:46, 28 March 2013
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	===Part 6: Preparation for next time===		===Part 6: Preparation for next time===

-	Next time, you will lyse your bacterial samples to release their proteins, and run these out on a protein gel. In order to compare the amount of protein in the -IPTG versus +IPTG samples, you would like to normalize by the number of cells. At ~~this point~~, you may have only three samples ready (-IPTG only), or you may have all six. In either case, measure the OD<sub>600</sub> of a 1:10 dilution of cells for each finished sample (for -IPTG you have done so already), and write this number down in your notebook and on today's [[Talk:20.109(~~S10~~):Induce_protein_and_evaluate_DNA_(Day5)\| Talk]] page. Then spin down the cells and aspirate the supernatant. Give the cell pellets to the teaching faculty; they will be stored frozen. (Be sure to make a 2X pellet for the +IPTG samples.)	+	Next time, you will lyse your bacterial samples to release their proteins, and prepare to run these out on a protein gel. In order to compare the amount of protein in the -IPTG versus +IPTG samples, you would like to normalize by the number of cells. At the end of today, you may have only three samples ready (-IPTG only), or you may have all six. In either case, measure the OD<sub>600</sub> of a 1:10 dilution of cells for each finished sample (actually, for the -IPTG samples you have done so already), and write this number down in your notebook and on today's [[Talk:20.109(S13):Induce_protein_and_evaluate_DNA_(Day5)\| Talk]] page. Then spin down the cells and aspirate the supernatant. Give the cell pellets to the teaching faculty; they will be stored frozen at -20 °C. (Be sure to make a 2X pellet for the +IPTG samples.)

	==For next time==		==For next time==

AgiStachowiak: /* Part 5: Cell observation and collection */

AgiStachowiak — Thu, 28 Mar 2013 17:45:16 GMT

Part 5: Cell observation and collection

←Older revision		Revision as of 17:45, 28 March 2013
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	#After ~2-3 hours, you will pour 1.5 mL from each tube (from Part 1) into a labeled eppendorf, then spin for 1 min. at maximum speed. Save the other 3 mL!		#After ~2-3 hours, you will pour 1.5 mL from each tube (from Part 1) into a labeled eppendorf, then spin for 1 min. at maximum speed. Save the other 3 mL!
		+	#*A minimum of 2.5 hours is best, assuming that timing doesn't take you past 4:45 pm.
	#Aspirate the supernatant from each eppendorf, using a fresh yellow pipet tip on the end of the glass pipet each time.		#Aspirate the supernatant from each eppendorf, using a fresh yellow pipet tip on the end of the glass pipet each time.
	#Observe the color of each of your pellets, and compare to the examples on today's [[Talk:20.109(S12):Induce_protein_and_evaluate_DNA_(Day5)\| Talk]] page. If the wild-type and both mutant pellets all appear yellow-greenish to the eye, proceed as follows:		#Observe the color of each of your pellets, and compare to the examples on today's [[Talk:20.109(S12):Induce_protein_and_evaluate_DNA_(Day5)\| Talk]] page. If the wild-type and both mutant pellets all appear yellow-greenish to the eye, proceed as follows:

AgiStachowiak: /* Part 4: Observe mutant colonies */

AgiStachowiak — Thu, 28 Mar 2013 17:44:36 GMT

Part 4: Observe mutant colonies

←Older revision		Revision as of 17:44, 28 March 2013
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	===Part 4: Observe mutant colonies===		===Part 4: Observe mutant colonies===

-	Last time you transformed BL21(DE3) cells with three different plasmids (two candidates for the X#Z mutant, and one ~~M124S~~/E67K/T79P clone). Compare the relative colony formation of cells carrying the different plasmids. If all the plates have dense cell growth, there is no need for you to get an exact colony count; just do your best to get a relative estimate, and describe any findings in your notebook.	+	Last time you transformed BL21(DE3) cells with three different plasmids (two candidates for the X#Z mutant, and one D24H/E67K/T79P/M124S clone). Compare the relative colony formation of cells carrying the different plasmids. If all the plates have dense cell growth, there is no need for you to get an exact colony count; just do your best to get a relative estimate, and describe any findings in your notebook.

	===Part 5: Cell observation and collection===		===Part 5: Cell observation and collection===

AgiStachowiak: /* Part 3: Analyze sequence data */

AgiStachowiak — Thu, 28 Mar 2013 17:44:05 GMT

Part 3: Analyze sequence data

←Older revision		Revision as of 17:44, 28 March 2013
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	Select order date 04-04-2012 for T/R results and 04-05-2012 for W/F results. You will all begin with the T/R link, which includes data for the three positive controls; start by analyzing the one you are working with, then move on to your unique mutant. The quickest way to start working with your data is to follow the "View" link under the Seq File heading. For ambiguous data, you may want to look directly at the Trace File as well.		Select order date 04-04-2012 for T/R results and 04-05-2012 for W/F results. You will all begin with the T/R link, which includes data for the three positive controls; start by analyzing the one you are working with, then move on to your unique mutant. The quickest way to start working with your data is to follow the "View" link under the Seq File heading. For ambiguous data, you may want to look directly at the Trace File as well.

-	Recall from Module 1 that you can align your sequencing data with ~~the~~ known inverse pericam ~~sequence~~ and the differences will be quickly identified. There are several web-based programs for aligning sequences and still more programs that can be purchased. The steps for using one web-based tool are sketched below. ~~You may use~~ CLUSTAL ~~(introduced to you in Module 1) if you prefer~~.	+	Recall from Module 1 that you can align your sequencing data with a known sequence, in this case inverse pericam, and the differences will be quickly identified. There are several web-based programs for aligning sequences and still more programs that can be purchased. The steps for using one web-based tool, the same one you used in Module 1, are sketched below. Another popular alignment program is [http://www.clustal.org CLUSTAL].

	<b>Align with "bl2seq" from [http://www.ncbi.nlm.nih.gov/ NCBI]</b>		<b>Align with "bl2seq" from [http://www.ncbi.nlm.nih.gov/ NCBI]</b>

AgiStachowiak: /* Part 3: Analyze sequence data */

AgiStachowiak — Thu, 28 Mar 2013 16:28:57 GMT

Part 3: Analyze sequence data

←Older revision		Revision as of 16:28, 28 March 2013
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	===Part 3: Analyze sequence data===		===Part 3: Analyze sequence data===

-	Your goal today is to analyze the sequencing data for three samples - two independent colonies from your X#Z mutant, and one ~~positive control~~ clone for practice - and then decide which colony to proceed with for the X#Z mutant.	+	Your goal today is to analyze the sequencing data for three samples - two independent colonies from your X#Z mutant, and one reference mutant clone for practice - and then decide which colony to proceed with for the X#Z mutant.

-	You will want to have the [[Media: S12-M2-20109_pRSET-IPC.gb\| pRSET-IPC ApE file]] handy, and to mark and/or note down the expected location of your mutation before proceeding. (Just compare to your annotation of the IPC alone ApE file that you prepared on Day 1 of the module.) The data from Genewiz is available at [http://genewiz.com this link]. Choose the "Login" link and then use "astachow@mit.edu" and "be20109" to log in. At the bottom right should be a link to download your sequencing results. Select order date 04-04-2012 for T/R results and 04-05-2012 for W/F results. You will all begin with the T/R link, which includes data for the three positive controls; start by analyzing the one you are working with, then move on to your unique mutant. The quickest way to start working with your data is to follow the "View" link under the Seq File heading. For ambiguous data, you may want to look directly at the Trace File as well.	+	<font color=red>Update sequencing info</font color>
		+	You will want to have the [[Media: S12-M2-20109_pRSET-IPC.gb\| pRSET-IPC ApE file]] handy, and to mark and/or note down the expected location of your mutation before proceeding. (Just compare to your annotation of the IPC alone ApE file that you prepared on Day 1 of the module.) The data from Genewiz is available at [http://genewiz.com this link]. Choose the "Login" link and then use "astachow@mit.edu" and "be20109" to log in. At the bottom right should be a link to download your sequencing results.

-	~~Rather than look through~~ the ~~sequence~~ to ~~magically find~~ the ~~relevant portion~~, you can align ~~the~~ data ~~you just got~~ with the ~~standard~~ inverse pericam sequence and the differences will be quickly identified. There are several web-based programs for aligning sequences and still more programs that can be purchased. The steps for using one web-based tool are sketched below. You may use CLUSTAL (introduced to you in Module 1) if you prefer.	+	Select order date 04-04-2012 for T/R results and 04-05-2012 for W/F results. You will all begin with the T/R link, which includes data for the three positive controls; start by analyzing the one you are working with, then move on to your unique mutant. The quickest way to start working with your data is to follow the "View" link under the Seq File heading. For ambiguous data, you may want to look directly at the Trace File as well.
		+
		+	Recall from Module 1 that you can align your sequencing data with the known inverse pericam sequence and the differences will be quickly identified. There are several web-based programs for aligning sequences and still more programs that can be purchased. The steps for using one web-based tool are sketched below. You may use CLUSTAL (introduced to you in Module 1) if you prefer.

	<b>Align with "bl2seq" from [http://www.ncbi.nlm.nih.gov/ NCBI]</b>		<b>Align with "bl2seq" from [http://www.ncbi.nlm.nih.gov/ NCBI]</b>

AgiStachowiak: /* Part 2: Run diagnostic gel */

AgiStachowiak — Thu, 28 Mar 2013 16:26:47 GMT

Part 2: Run diagnostic gel

←Older revision		Revision as of 16:26, 28 March 2013
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	===Part 2: Run diagnostic gel===		===Part 2: Run diagnostic gel===

-	The scheme below assumes that both Digest 1 (D1, used to analyze the positive control mutant) and Digest 2 (D2, used to analyze your X#Z mutant candidates) use only one enzyme. If you are doing double-digests and need to run single enzyme controls, hopefully you spoke to the teaching faculty about this last time. Load your samples on a 1% agarose gel in the following order, using 10 μL per ladder and 20 μL per plasmid:	+	The scheme below assumes that both Digest 1 (D1, used to analyze the positive control mutant) and Digest 2 (D2, used to analyze your X#Z mutant candidates) use only one enzyme. If you are doing double-digests and need to run single enzyme controls, hopefully you spoke to the teaching faculty about this plan last time. Load your samples on a 1% agarose gel in the following order, using 10 μL per ladder and 20 μL per plasmid:

	<center>		<center>
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	</center>		</center>

-	Once all the samples are loaded, the power will be applied (100 V for 45 minutes) and ~~the gel~~ will ~~be photographed~~. When the gel is ready, you will compare the band sizes in the photograph with the expected band sizes that you previously calculated. In the meantime, you can analyze your sequence data.	+	Once all the samples are loaded, the power will be applied (100 V for 45 minutes), and then you will photograph your gels.
		+
		+	When the gel is ready, you will compare the band sizes in the photograph with the expected band sizes that you previously calculated. In the meantime, you can analyze your sequence data.
		+
		+	Before you leave today, '''please upload your gel image according to the filenames on today's Talk page'''. The code should automatically display your image on the Talk page after the upload is complete.

	===Part 3: Analyze sequence data===		===Part 3: Analyze sequence data===