IGEM 2009 notebook - Revision history

Roclemente: /* Roclemente 15:12, 29 June 2009 (EDT) */

2009-06-29T20:15:54Z

‎Roclemente 15:12, 29 June 2009 (EDT)

Roclemente: /* Roclemente 15:12, 26 June 2009 (EDT) */

2009-06-29T20:15:39Z

‎Roclemente 15:12, 26 June 2009 (EDT)

Roclemente: /* Roclemente 15:12, 29 June 2009 (EDT) */

2009-06-29T20:15:17Z

‎Roclemente 15:12, 29 June 2009 (EDT)

Roclemente at 19:12, 29 June 2009

2009-06-29T19:12:28Z

Roclemente: /* Roclemente 11:26, 25 June 2009 (EDT) */

2009-06-29T19:03:40Z

‎Roclemente 11:26, 25 June 2009 (EDT)

Roclemente: /* Roclemente 11:26, 26 June 2009 (EDT) */

2009-06-26T15:36:48Z

‎Roclemente 11:26, 26 June 2009 (EDT)

Roclemente at 15:26, 26 June 2009

2009-06-26T15:26:58Z

Roclemente: /* Roclemente 15:09, 24 June 2009 (EDT) */

2009-06-26T15:24:56Z

‎Roclemente 15:09, 24 June 2009 (EDT)

Roclemente: /* Roclemente 15:09, 24 June 2009 (EDT) */

2009-06-24T19:29:53Z

‎Roclemente 15:09, 24 June 2009 (EDT)

Roclemente at 19:09, 24 June 2009

2009-06-24T19:09:01Z

← Older revision		Revision as of 20:15, 29 June 2009
Line 517:		Line 517:

	In trying to figure out how much tRNA I wanted to digest, I thought about how big the tRNA insrt would be relative to the entire tRNA part in the plasmid. This would be minimal, so I decided to digest 2000 ng of tRNA to account for the DNA I will presumably lose along the way. I would have a final volume of 20 uL.		In trying to figure out how much tRNA I wanted to digest, I thought about how big the tRNA insrt would be relative to the entire tRNA part in the plasmid. This would be minimal, so I decided to digest 2000 ng of tRNA to account for the DNA I will presumably lose along the way. I would have a final volume of 20 uL.
−
−	Since the pBad vector that we want is a much larger percentage of the digested product, I wouldn't need to digest as much, but I still wanted to have enough for the whole group of us to use eventually. I chose to digest 1000 ng of the pBad part (I13453).
−
−	~~== [[User:Roclemente\|Roclemente]] 15:12, 29 June 2009 (EDT) ==~~
−
−	I miniprepped 120 uL of tRNA_CUAGU. This tRNA had a concentration of 17.1 ng/uL. I left aside one liquid culture of this tRNA so that I could keep these cells in the freezer. I added my tRNA to the registry as the part number K199001 and under the name "CUAGU tRNA gene." I placed the cells, mixed with glycogen, in the freezer in box 3-4 at position 81.
−
−	Even though I hadn't yet verified my RFP and Tet sequences through DNA sequencing, I added these pars to the registry because I would eventually have these parts afer sequencing. My RFP CUAGU is now part K199003 and my Tet CUAGU is part K199004.
−
−	We, as an iGEM team, decided to use pBad as our promoter for each reporter gene and tRNA. We need to put the pBad promoter and RBS in front of both reporter genes. We need to put just the pBad in front of the tRNAs. We don't need an RBS for the tRNAs since we aren't intending to translate it. Since tRNA is longer than the pBAD promoter, we will be inserting our tRNA into the pBad vector. Therefore, I want to digest my CUAGU tRNA with XbaI and PstI. and I want to digest my CUAGU reporter genes with SpeI and PstI.
−
−	In trying to figure out how much tRNA I wanted to digest, I thought about how big the tRNA insrt would be relative to the entire tRNA part in the plasmid. This would be minimal, so I decided to digest 2000 ng of tRNA to account for the DNA I will presumably lose along the way. I would have a final volume of 20 uL.

	Since the pBad vector that we want is a much larger percentage of the digested product, I wouldn't need to digest as much, but I still wanted to have enough for the whole group of us to use eventually. I chose to digest 1000 ng of the pBad part (I13453).		Since the pBad vector that we want is a much larger percentage of the digested product, I wouldn't need to digest as much, but I still wanted to have enough for the whole group of us to use eventually. I chose to digest 1000 ng of the pBad part (I13453).

← Older revision		Revision as of 20:15, 29 June 2009
Line 509:		Line 509:

	[[Image:Sequencing.png]]		[[Image:Sequencing.png]]
		+
		+	I miniprepped 120 uL of tRNA_CUAGU. This tRNA had a concentration of 17.1 ng/uL. I left aside one liquid culture of this tRNA so that I could keep these cells in the freezer. I added my tRNA to the registry as the part number K199001 and under the name "CUAGU tRNA gene." I placed the cells, mixed with glycogen, in the freezer in box 3-4 at position 81.
		+
		+	Even though I hadn't yet verified my RFP and Tet sequences through DNA sequencing, I added these pars to the registry because I would eventually have these parts afer sequencing. My RFP CUAGU is now part K199003 and my Tet CUAGU is part K199004.
		+
		+	We, as an iGEM team, decided to use pBad as our promoter for each reporter gene and tRNA. We need to put the pBad promoter and RBS in front of both reporter genes. We need to put just the pBad in front of the tRNAs. We don't need an RBS for the tRNAs since we aren't intending to translate it. Since tRNA is longer than the pBAD promoter, we will be inserting our tRNA into the pBad vector. Therefore, I want to digest my CUAGU tRNA with XbaI and PstI. and I want to digest my CUAGU reporter genes with SpeI and PstI.
		+
		+	In trying to figure out how much tRNA I wanted to digest, I thought about how big the tRNA insrt would be relative to the entire tRNA part in the plasmid. This would be minimal, so I decided to digest 2000 ng of tRNA to account for the DNA I will presumably lose along the way. I would have a final volume of 20 uL.
		+
		+	Since the pBad vector that we want is a much larger percentage of the digested product, I wouldn't need to digest as much, but I still wanted to have enough for the whole group of us to use eventually. I chose to digest 1000 ng of the pBad part (I13453).

	== [[User:Roclemente\|Roclemente]] 15:12, 29 June 2009 (EDT) ==		== [[User:Roclemente\|Roclemente]] 15:12, 29 June 2009 (EDT) ==

@@ Line 504: / Line 504: @@
 There was a mysterious longer band in the 8th RFP, but other than that, the rest of my RFPs looked good. I will send these dried down samples in for sequencing.
-== [[User:Roclemente|Roclemente]] 15:12, 29 June 2009 (EDT) ==
+== [[User:Roclemente|Roclemente]] 15:12, 26 June 2009 (EDT) ==
 I needed 120 ng of each piece of DNA I wanted to confirm by sequencing. I miniprepped each of the DNA sequences that I verified through running a gel. Since my minipreps were too concenrated for use on the NanoDrop, I mixed 1 uL of my DNA with 5 uL water. I blanked the Nanodrop with the same distilled water and got the following concentrations. I multiplied this concentration by 5. Then, I divided 120 ng by this concentration in order to find out how much of my orinigal miniprepped DNA I needed to dry down and send for sequencing.

← Older revision		Revision as of 19:12, 29 June 2009
Line 502:		Line 502:
	[[Image:2009-06-25 15hr 12min.jpg\|thumb\|none\|300px\|1.2% gel. Lane 1: MW marker. Lanes 2-11: RFP_CUAGU 1-10]]		[[Image:2009-06-25 15hr 12min.jpg\|thumb\|none\|300px\|1.2% gel. Lane 1: MW marker. Lanes 2-11: RFP_CUAGU 1-10]]

−	There was a mysterious longer band in the 8th RFP, but other than that, the rest of my RFPs looked good. I ~~sent~~ these dried down samples in for sequencing.	+	There was a mysterious longer band in the 8th RFP, but other than that, the rest of my RFPs looked good. I will send these dried down samples in for sequencing.
		+
		+	== [[User:Roclemente\|Roclemente]] 15:12, 29 June 2009 (EDT) ==
		+
		+	I needed 120 ng of each piece of DNA I wanted to confirm by sequencing. I miniprepped each of the DNA sequences that I verified through running a gel. Since my minipreps were too concenrated for use on the NanoDrop, I mixed 1 uL of my DNA with 5 uL water. I blanked the Nanodrop with the same distilled water and got the following concentrations. I multiplied this concentration by 5. Then, I divided 120 ng by this concentration in order to find out how much of my orinigal miniprepped DNA I needed to dry down and send for sequencing.
		+
		+	[[Image:Sequencing.png]]
		+
		+	== [[User:Roclemente\|Roclemente]] 15:12, 29 June 2009 (EDT) ==
		+
		+	I miniprepped 120 uL of tRNA_CUAGU. This tRNA had a concentration of 17.1 ng/uL. I left aside one liquid culture of this tRNA so that I could keep these cells in the freezer. I added my tRNA to the registry as the part number K199001 and under the name "CUAGU tRNA gene." I placed the cells, mixed with glycogen, in the freezer in box 3-4 at position 81.
		+
		+	Even though I hadn't yet verified my RFP and Tet sequences through DNA sequencing, I added these pars to the registry because I would eventually have these parts afer sequencing. My RFP CUAGU is now part K199003 and my Tet CUAGU is part K199004.
		+
		+	We, as an iGEM team, decided to use pBad as our promoter for each reporter gene and tRNA. We need to put the pBad promoter and RBS in front of both reporter genes. We need to put just the pBad in front of the tRNAs. We don't need an RBS for the tRNAs since we aren't intending to translate it. Since tRNA is longer than the pBAD promoter, we will be inserting our tRNA into the pBad vector. Therefore, I want to digest my CUAGU tRNA with XbaI and PstI. and I want to digest my CUAGU reporter genes with SpeI and PstI.
		+
		+	In trying to figure out how much tRNA I wanted to digest, I thought about how big the tRNA insrt would be relative to the entire tRNA part in the plasmid. This would be minimal, so I decided to digest 2000 ng of tRNA to account for the DNA I will presumably lose along the way. I would have a final volume of 20 uL.
		+
		+	Since the pBad vector that we want is a much larger percentage of the digested product, I wouldn't need to digest as much, but I still wanted to have enough for the whole group of us to use eventually. I chose to digest 1000 ng of the pBad part (I13453).

← Older revision		Revision as of 19:03, 29 June 2009
Line 502:		Line 502:
	[[Image:2009-06-25 15hr 12min.jpg\|thumb\|none\|300px\|1.2% gel. Lane 1: MW marker. Lanes 2-11: RFP_CUAGU 1-10]]		[[Image:2009-06-25 15hr 12min.jpg\|thumb\|none\|300px\|1.2% gel. Lane 1: MW marker. Lanes 2-11: RFP_CUAGU 1-10]]

−	I s	+	There was a mysterious longer band in the 8th RFP, but other than that, the rest of my RFPs looked good. I sent these dried down samples in for sequencing.

← Older revision		Revision as of 15:36, 26 June 2009
Line 490:		Line 490:
	The sequences in lanes 2-4 and lanes 6-12 all seem to be about 985 bp so I went on to prepare these RFPs for sequencing. I tossed the RFP minipreps used in lanes 5 and 13.		The sequences in lanes 2-4 and lanes 6-12 all seem to be about 985 bp so I went on to prepare these RFPs for sequencing. I tossed the RFP minipreps used in lanes 5 and 13.

−	== [[User:Roclemente\|Roclemente]] 11:26, 26 June 2009 (EDT) ==	+	== [[User:Roclemente\|Roclemente]] 11:26, 25 June 2009 (EDT) ==
		+
		+	I continued catching my RFPs up to speed with my Tet FMLs by digesting my 10 tubes of RFP FMLs. We want to digest the RFPs for the same reason we digested our Tets: we want the difference between whether or not our 5mer was inserted to be more apparent. If the 5mer was inserted, the length we would see on a gel should be 443 bp (this includes the prefix, 5mer, and the part of the RFP gene before the NcoI site). If the 5mer wasn't inserted, the length we would see on a gel should be 645 bp (this includes the promoter, RBS, and the part of the RFP gene before the NcoI site).
		+
		+	For my digestion, I made a master mix enough for 12 digestions even though I was only digesting 10 tubes of DNA:
		+
		+	[[Image:Rfpgel.png]]
		+
		+	I let this digestion incubate for 1 hour and no more since I just needed to see the bands for verification rather than purification. Since the optimal gel for a 443 bp sequence is 1.3% and the optimal gel for a 645 bp sequence is 1.1% gel, I decided to use a 1.2% gel:
		+
		+	[[Image:2009-06-25 15hr 12min.jpg\|thumb\|none\|300px\|1.2% gel. Lane 1: MW marker. Lanes 2-11: RFP_CUAGU 1-10]]
		+
		+	I s

← Older revision		Revision as of 15:26, 26 June 2009
Line 489:		Line 489:

	The sequences in lanes 2-4 and lanes 6-12 all seem to be about 985 bp so I went on to prepare these RFPs for sequencing. I tossed the RFP minipreps used in lanes 5 and 13.		The sequences in lanes 2-4 and lanes 6-12 all seem to be about 985 bp so I went on to prepare these RFPs for sequencing. I tossed the RFP minipreps used in lanes 5 and 13.
		+
		+	== [[User:Roclemente\|Roclemente]] 11:26, 26 June 2009 (EDT) ==

@@ Line 474: / Line 474: @@
 Instead of digesting the Tet with EcoR1 and Pst1, we decided to digest it with EcoR1 and BamH1. The only difference between the Tet that would have ligated to itself and the Tet inserted with the FML is the length of the FML. Since the length of the FML is very small compared to the length of the entire gene up to Pst1, we would be able to visualize a larger difference in size on a gel if this FML length made up a larger percentage of the sequence we will see. This sequence we would have if we cut our plasmids with BamH1 would be about 800 bp smaller than the sequence we would have seen if we would have cut our plasmids with Pst1.
-i used the following mix for my digestion for each of my 7 Tet_CUAGU tubes:
+I used the following mix for my digestion for each of my 7 Tet_CUAGU tubes:
 [[Image:Tetcuagudigestion.png]]

← Older revision		Revision as of 19:29, 24 June 2009
Line 468:		Line 468:
	I checked on my RFP and negative control RFP transformations yesterday. My RFP plate had many colonies, but my negative control plate had a lot of colonies too. Nevertheless, I prepared colony PCRs for 11 white colonies and 1 red colony using the [http://www.bio.davidson.edu/courses/Molbio/Protocols/ColonyPCR_Screening.html Colony PCR to Screen for Successful Ligations] protocol on the Davidson Lab Protocols link. I made the following master mix, enough for 14 colony PCRs so that I would have extra just in case:		I checked on my RFP and negative control RFP transformations yesterday. My RFP plate had many colonies, but my negative control plate had a lot of colonies too. Nevertheless, I prepared colony PCRs for 11 white colonies and 1 red colony using the [http://www.bio.davidson.edu/courses/Molbio/Protocols/ColonyPCR_Screening.html Colony PCR to Screen for Successful Ligations] protocol on the Davidson Lab Protocols link. I made the following master mix, enough for 14 colony PCRs so that I would have extra just in case:

−	[[Image:~~Rfpcolonypcr~~.png]]	+	[[Image:Rfp2colonypcr.png]]
		+
		+	Afterwards, I wanted to miniprep my Tet_CUAGU cells in preparation to digest them and figure out which plasmids actually contain the FML and did not simply ligate to itself. First, I plated each of the 7 tubes of cells to save these cells. This way, I could use all the rest of my overnight culture for the miniprep.
		+
		+	Instead of digesting the Tet with EcoR1 and Pst1, we decided to digest it with EcoR1 and BamH1. The only difference between the Tet that would have ligated to itself and the Tet inserted with the FML is the length of the FML. Since the length of the FML is very small compared to the length of the entire gene up to Pst1, we would be able to visualize a larger difference in size on a gel if this FML length made up a larger percentage of the sequence we will see. This sequence we would have if we cut our plasmids with BamH1 would be about 800 bp smaller than the sequence we would have seen if we would have cut our plasmids with Pst1.
		+
		+	i used the following mix for my digestion for each of my 7 Tet_CUAGU tubes:
		+
		+	[[Image:Tetcuagudigestion.png]]

← Older revision		Revision as of 19:09, 24 June 2009
Line 463:		Line 463:

	We can only eliminate the DNA for the PCR bands that look about 1100 bp long. However, if they are about 1450 bp long, they could have either ligated to itself and included the whole gene or ligated with the 5mer. Thankfully, none of my 6 Tet PCR screens look any less than about 1450 bp so I added more LB+Amp to these liquid cultures and will allow them to culture overnight. I did the same with my negative control.		We can only eliminate the DNA for the PCR bands that look about 1100 bp long. However, if they are about 1450 bp long, they could have either ligated to itself and included the whole gene or ligated with the 5mer. Thankfully, none of my 6 Tet PCR screens look any less than about 1450 bp so I added more LB+Amp to these liquid cultures and will allow them to culture overnight. I did the same with my negative control.
		+
		+	== [[User:Roclemente\|Roclemente]] 15:09, 24 June 2009 (EDT) ==
		+
		+	I checked on my RFP and negative control RFP transformations yesterday. My RFP plate had many colonies, but my negative control plate had a lot of colonies too. Nevertheless, I prepared colony PCRs for 11 white colonies and 1 red colony using the [http://www.bio.davidson.edu/courses/Molbio/Protocols/ColonyPCR_Screening.html Colony PCR to Screen for Successful Ligations] protocol on the Davidson Lab Protocols link. I made the following master mix, enough for 14 colony PCRs so that I would have extra just in case:
		+
		+	[[Image:Rfpcolonypcr.png]]