User:Kchang17/notes - Revision history

Kchang17: /* Induction */

Kchang17 — Fri, 13 Oct 2006 03:49:29 GMT

Induction

←Older revision		Revision as of 03:49, 13 October 2006
Line 729:		Line 729:
	*added 1.6e6 cells to 25ml experimental cultures (0%, 1e-4%)		*added 1.6e6 cells to 25ml experimental cultures (0%, 1e-4%)
	**this is about 10x the library sizes		**this is about 10x the library sizes
		+
		+	===MOFLO===
		+	*fire alarm at flow cytometry center, no data :(
		+	*libraries didn't seem to have working clones

Kchang17: /* Day 4 (Aug. 25) */

Kchang17 — Thu, 31 Aug 2006 19:11:42 GMT

Day 4 (Aug. 25)

←Older revision		Revision as of 19:11, 31 August 2006
Line 618:		Line 618:
	**Q04740 low in/high out labeled L.Q04740.1.0		**Q04740 low in/high out labeled L.Q04740.1.0
	**Q04740 high in/low out labeled L.Q04740.1.1		**Q04740 high in/low out labeled L.Q04740.1.1
-	**Q04400 low in/high out labeled L.~~Q04740~~.1.0	+	**Q04400 low in/high out labeled L.Q04400.1.0

	===Day 5 (Aug. 28)===		===Day 5 (Aug. 28)===

Kchang17: /* Library sizes */

Kchang17 — Wed, 30 Aug 2006 23:50:36 GMT

Library sizes

←Older revision		Revision as of 23:50, 30 August 2006
Line 678:		Line 678:
	*added 10ml M9+AG, grew overnight ~12.5 hrs		*added 10ml M9+AG, grew overnight ~12.5 hrs

-	===Library sizes===	+	===Library sizes (August 30)===
	{\| border=1		{\| border=1
	\|+<b>Plating Results</b>		\|+<b>Plating Results</b>

Kchang17: /* Restriction Digests */

Kchang17 — Wed, 30 Aug 2006 23:50:21 GMT

Restriction Digests

←Older revision		Revision as of 23:50, 30 August 2006
Line 648:		Line 648:
	*PCR cleaned the rest into 30 ul EB		*PCR cleaned the rest into 30 ul EB

-	===Restriction Digests===	+	===Restriction Digests (August 29)===
	*20min, 37 C restriction digests on libraries (XP), heat inactivation		*20min, 37 C restriction digests on libraries (XP), heat inactivation
	*PCR cleanup & elution into 30ul		*PCR cleanup & elution into 30ul

Kchang17: /* Library sizes */

Kchang17 — Wed, 30 Aug 2006 23:49:27 GMT

Library sizes

←Older revision		Revision as of 23:49, 30 August 2006
Line 701:		Line 701:
	\| 512x8 \|\| 156 \|\| 1.6e5		\| 512x8 \|\| 156 \|\| 1.6e5
	\|}		\|}
		+
		+	===Induction===
		+	*spun down, resuspended in M9+AG
		+	{\| border=1
		+	\|+<b>Plating Results</b>
		+	! library !! OD
		+	\|-
		+	! P8/C2002
		+	\| 0.53
		+	\|-
		+	! P9/C2002
		+	\| 0.45
		+	\|-
		+	! R2000/C2002
		+	\| 0.49
		+	\|-
		+	! P8/C2003
		+	\| 0.43
		+	\|-
		+	! P9/C2003
		+	\| 0.43
		+	\|-
		+	! R2000/C2003
		+	\| 0.34
		+	\|}
		+	*made glycerols (labeled with L.RS.P8/P9/R2000 and B0032.C2002.B0015/B0030.C2003.B0015)
		+	*added 1.6e6 cells to 25ml experimental cultures (0%, 1e-4%)
		+	**this is about 10x the library sizes

Kchang17: /* Day 6 (Aug. 29): MoFlo */

Kchang17 — Wed, 30 Aug 2006 23:45:48 GMT

Day 6 (Aug. 29): MoFlo

←Older revision		Revision as of 23:45, 30 August 2006
Line 633:		Line 633:
	**7268 cells		**7268 cells
	*grew in 1 ml M9+AG until next day		*grew in 1 ml M9+AG until next day
		+
		+	===Day 7 (Aug. 30)===
		+	*added 5 ml to the sublibrary, grew ~7.5 hrs
		+	**OD = 0.83
		+	*made 1 glycerol: L.Q04400.1.0.1
		+	*added 1.4e6 cells to 25ml experimental cultures (0%, 1e-4%)

	==August 28, 2006: Reshma's promoter libraries==		==August 28, 2006: Reshma's promoter libraries==

Kchang17: /* Ligation */

Kchang17 — Wed, 30 Aug 2006 19:49:55 GMT

Ligation

←Older revision		Revision as of 19:49, 30 August 2006
Line 662:		Line 662:
	**save 1ul for gel analysis		**save 1ul for gel analysis
	*dialysis 30min		*dialysis 30min
-	[[Image:August30_2006_gel.jpg\|thumb\|left\|300px\|comparisons of restriction digests, ligations, and dialyzed ligations (1ul volumes of each); left to right: C2002 backbone (SP), C2003 backbone (SP), P8 libary (XP), P9 library (XP), R2000 library (XP), P8/C2002 ligation, P8/C2002 dialysis, P9/C2002 ligation, P9/C2002 dialysis, R2000/C2002 ligation, R2000/C2002 dialysis, P8/C2003 ligation, P8/C2003 dialysis, P9/C2003 ligation, P9/C2003 dialysis, R2000/C2003 ligation, R2000/C2003 dialysis]] [[Image:August30_2006_gel_bright.jpg\|thumb\|none\|300px\|same gel with longer UV exposure to help see the insert better]]~~<br><br><br><br><br><br><br><br><br>~~<br><br><br><br><br><br>	+	[[Image:August30_2006_gel.jpg\|thumb\|left\|300px\|comparisons of restriction digests, ligations, and dialyzed ligations (1ul volumes of each); left to right: C2002 backbone (SP), C2003 backbone (SP), P8 libary (XP), P9 library (XP), R2000 library (XP), P8/C2002 ligation, P8/C2002 dialysis, P9/C2002 ligation, P9/C2002 dialysis, R2000/C2002 ligation, R2000/C2002 dialysis, P8/C2003 ligation, P8/C2003 dialysis, P9/C2003 ligation, P9/C2003 dialysis, R2000/C2003 ligation, R2000/C2003 dialysis]] [[Image:August30_2006_gel_bright.jpg\|thumb\|none\|300px\|same gel with longer UV exposure to help see the insert better]]<br><br><br><br><br><br>

	===Transformation===		===Transformation===

Kchang17: /* August 28, 2006: Reshma's promoter libraries */

Kchang17 — Wed, 30 Aug 2006 19:46:42 GMT

August 28, 2006: Reshma's promoter libraries

←Older revision		Revision as of 19:46, 30 August 2006
Line 639:		Line 639:
	**L.RS.P9		**L.RS.P9
	**L.RS.R2000		**L.RS.R2000
-	*saved 1 ul (out of 100 ul PCR reaction) for gel analysis	+	*saved 1 ul (out of 100 ul PCR reaction) for gel analysis [[Image:August29_2006_ds.jpg\|thumb\|none\|150px\|1ul of PCR reaction compared next to 1ul of 1:40 dilution of original single-stranded library (lanes alternate)]]
	*PCR cleaned the rest into 30 ul EB		*PCR cleaned the rest into 30 ul EB

Line 652:		Line 652:
	*B0032.C2002.B0015 in SP1.0 = 319.9ng/ul 30ul		*B0032.C2002.B0015 in SP1.0 = 319.9ng/ul 30ul
	*B0030.C2003.B0015 in SP1.0 = 300.5ng/ul 30ul		*B0030.C2003.B0015 in SP1.0 = 300.5ng/ul 30ul
		+
		+	*used 1ul of these samples for gel analysis

	===Ligation===		===Ligation===
Line 660:		Line 662:
	**save 1ul for gel analysis		**save 1ul for gel analysis
	*dialysis 30min		*dialysis 30min
		+	[[Image:August30_2006_gel.jpg\|thumb\|left\|300px\|comparisons of restriction digests, ligations, and dialyzed ligations (1ul volumes of each); left to right: C2002 backbone (SP), C2003 backbone (SP), P8 libary (XP), P9 library (XP), R2000 library (XP), P8/C2002 ligation, P8/C2002 dialysis, P9/C2002 ligation, P9/C2002 dialysis, R2000/C2002 ligation, R2000/C2002 dialysis, P8/C2003 ligation, P8/C2003 dialysis, P9/C2003 ligation, P9/C2003 dialysis, R2000/C2003 ligation, R2000/C2003 dialysis]] [[Image:August30_2006_gel_bright.jpg\|thumb\|none\|300px\|same gel with longer UV exposure to help see the insert better]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>

	===Transformation===		===Transformation===

Kchang17: /* Library sizes */

Kchang17 — Wed, 30 Aug 2006 18:26:11 GMT

Library sizes

←Older revision		Revision as of 18:26, 30 August 2006
Line 675:		Line 675:
	\|-		\|-
	! P8/C2002		! P8/C2002
-	\| 1391x2 \|\| 305	+	\| 1391x2 \|\| 305 \|\| 1.1e5
	\|-		\|-
	! P9/C2002		! P9/C2002
-	\| 353x8 \|\| 185	+	\| 353x8 \|\| 185 \|\| 1.1e5
	\|-		\|-
	! R2000/C2002		! R2000/C2002
-	\| 411x8 \|\| 177	+	\| 411x8 \|\| 177 \|\| 1.3e5
	\|-		\|-
	! P8/C2003		! P8/C2003
-	\| 385x8 \|\| 178	+	\| 385x8 \|\| 178 \|\| 1.2e5
	\|-		\|-
	! P9/C2003		! P9/C2003
-	\| 515x8 \|\| 194	+	\| 515x8 \|\| 194 \|\| 1.6e5
	\|-		\|-
	! R2000/C2003		! R2000/C2003
-	\| 512x8 \|\| 156	+	\| 512x8 \|\| 156 \|\| 1.6e5
	\|}		\|}

Kchang17: /* Transformation */

Kchang17 — Wed, 30 Aug 2006 18:23:09 GMT

Transformation

←Older revision		Revision as of 18:23, 30 August 2006
Line 664:		Line 664:
	*electroporated 3ul, except for the one I messed up, did 6.3ul for that one		*electroporated 3ul, except for the one I messed up, did 6.3ul for that one
	*on ice, then incubate at 37 C for 1hr		*on ice, then incubate at 37 C for 1hr
		+	*plated:
		+	**LB+AG 1:40 dilution (25ul of transformation directly)
		+	**LB only 1:1e6 dilution
	*added 10ml M9+AG, grew overnight ~12.5 hrs		*added 10ml M9+AG, grew overnight ~12.5 hrs
		+
		+	===Library sizes===
		+	{\| border=1
		+	\|+<b>Plating Results</b>
		+	! library !! LB+AG !! LB only !! library size
		+	\|-
		+	! P8/C2002
		+	\| 1391x2 \|\| 305
		+	\|-
		+	! P9/C2002
		+	\| 353x8 \|\| 185
		+	\|-
		+	! R2000/C2002
		+	\| 411x8 \|\| 177
		+	\|-
		+	! P8/C2003
		+	\| 385x8 \|\| 178
		+	\|-
		+	! P9/C2003
		+	\| 515x8 \|\| 194
		+	\|-
		+	! R2000/C2003
		+	\| 512x8 \|\| 156
		+	\|}