User:Matthewmeisel - Revision history

ShawnDouglas: /* Other projects */

2006-07-18T01:16:50Z

Other projects

←Older revision		Revision as of 01:16, 18 July 2006
Line 68:		Line 68:

	My [http://www.freebiology.org/wiki/User:MatthewMeisel work] in Biophysics 101		My [http://www.freebiology.org/wiki/User:MatthewMeisel work] in Biophysics 101
		+
		+	==Lachrymosity==
		+	*[[IGEM:Harvard/2006/Why Apples make me cry \| Why Apples make me cry]] (feel free to contribute)

	==Contact info==		==Contact info==

	Email me: (my last name) at fas dot harvard dot edu		Email me: (my last name) at fas dot harvard dot edu

Matthewmeisel at 14:48, 13 July 2006

2006-07-13T14:48:05Z

←Older revision		Revision as of 14:48, 13 July 2006
Line 1:		Line 1:
-	~~Quick link~~: [[iGEM:Harvard/2006]]	+	[[iGEM:Harvard]] \| [[iGEM:Harvard/2006\|2006]]

	==Working file scrapbook==		==Working file scrapbook==

Matthewmeisel at 20:41, 10 July 2006

2006-07-10T20:41:27Z

←Older revision		Revision as of 20:41, 10 July 2006
Line 15:		Line 15:
	weekstart=14		weekstart=14
	</calendar>		</calendar>
-
-	~~===Week 2===~~
-
-	~~====Tue Jun 20====~~
-
-	~~Transformation of standard components~~
-	* cultured bacteria did grow overnight
-	* 1 mL of reserved to make glycerol stock:
-	~~# 666 {{ul}} of cells and medium and 666 {{ul}} of 50% glycerol solution in water~~
-	~~# stored at 80{{c}}~~
-	* extracted DNA from remaining 4 mL using the [[Miniprep/Qiagen_kit\|standard protocol]]:
-	~~#Cells and medium decanted into several 1.5 mL microcentrifuge tubes, centrifuged, and supernatant discarded.~~
-	~~#Resuspended pelleted bacterial cells in 250 µl Buffer P1 (w/ RNAse) (kept at 4 °C).~~
-	~~#Added 250 μl Buffer P2 and gently inverted the tube 4–6 times to mix. Waited 2 min.~~
-	~~#Added 350 μl Buffer N3 and inverted the tube immediately but gently 4–6 times. Solution became cloudy.~~
-	~~#Centrifuged for 10 min at 14,000 rpm. A compact white pellet formed.~~
-	~~#Combined the supernatants from step 4 in a single QIAprep spin column by decanting.~~
-	~~#Centrifuged for 60 s. Discarded the flow-through.~~
-	~~#Washed the QIAprep spin column with 0.5 ml Buffer PB and centrifuged for 60 s. Discarded the flow-through.~~
-	~~#Washed QIAprep spin column by with 0.75 ml Buffer PE and centrifuged for 60 s.~~
-	~~#Placed the QIAprep column in a clean 1.5 ml microcentrifuge tube. Eluted with 30 μl water to the center of each QIAprep spin column, let stand for 2 min, and centrifuged for 60 s.~~
-	* digested to verfiy that the insert was inserted
-	~~#The following ingredients were each added to a 1.5 ml centrifuge tube:~~
-	~~#*10 μl water~~
-	~~#*8 μl DNA from the previous step~~
-	~~#*2.5 μl 10x NEB buffer (#2)~~
-	~~#*2.5 μl 10x BSE~~
-	~~#*1.0 μl 1:1 diluted enzyme A (SpeI)~~
-	~~#*1.0 μl 1:1 diluted enzyme B (XbaI).~~
-	~~#Tubes were incubated at 37°C for 3 h.~~
-	~~#Tubes were incubated at 80°C for 15 min in order to inactivate the enzymes.~~
-	~~#10 {{ul}} of digest reaction and 10 {{ul}} water were mixed and run in a 1.2% agarose Invitrogen E-gel for 30 min. (ladder in lane 1, our sample in lane 4).~~
-
-	~~====Mon Jun 19====~~
-
-	~~Transformation of standard components~~
-	* Viewed plate from Thu, about a dozen colonies were flourescing brightly
-	* Selected one colony for amplification:
-	~~# 5 mL LB medium, 50 {{ul}} of 5 mg/mL ampicillin, and one colony into a culture tube~~
-	~~# Shaken at 18 h at 37{{c}}~~
-
-	~~===Week 1===~~
-
-	~~====Thu Jun 15====~~
-
-	~~Transformation of standard components~~
-	* ligation and transformation of promoter and GFP
-	* ligation protocol with the Roche Rapid DNA ligation kit:
-	# 6 {{ul}} of insert (E0241), 1 {{ul}} of plasmid (R0010 and backbone), and 3 {{ul}} of 1x DNA dilution buffer (vial 2) (enough buffer for total volume of 10 {{ul}}) into a microcentrifuge tube. (Would ordinarily use 2 {{ul}} of plasmid, but the plasmid concentration is about doubled because it combined bands from two gel lanes.)
-	~~# Mixed ligation buffer (vial 1), and added 10 {{ul}} to tube.~~
-	~~# Added 1 {{ul}} T4 DNA ligase (vial 3).~~
-	~~# Incubated on ice for 5 min.~~
-	* transformation:
-	~~# 30 {{ul}} competent cells each into three microcentrifuge tubes.~~
-	~~# Ligation mixture, 1 {{ul}} water (negative control), and 1 {{uL}} pUC 19 (positive control) added to respective tubes.~~
-	~~# Incubated the tubes on ice for 20 min.~~
-	~~# Heat shocked the tubes at 42°C for 30 s.~~
-	~~# Added 200 {{ul}} SOC to each tube.~~
-	~~# Incubated, with shaking, at 37°C for 1 h.~~
-	~~# Plated the mixture on carbomycin plates and incubated at 37°C for 24 h, then stored at 4°C.~~
-
-	~~====Wed Jun 14====~~
-
-	~~Transformation of standard components~~
-	* cultured bacteria did grow overnight
-	* 1 mL of each tube reserved to make glycerol stock:
-	~~# 666 {{ul}} of cells and medium and 666 {{ul}} of 50% glycerol solution in water~~
-	~~# stored at 80{{c}}~~
-	* extracted DNA from remaining 4 mL using the [[Miniprep/Qiagen_kit\|standard protocol]]:
-	~~#Cells and medium poured into 1.5 mL microcentrifuge tubes, centrifuged, and supernatant discarded.~~
-	~~#Resuspended pelleted bacterial cells in 250 µl Buffer P1 (w/ RNAse) (kept at 4 °C) and transfered to a microcentrifuge tube.~~
-	~~#Added 250 μl Buffer P2 and gently inverted the tube 4–6 times to mix. Waited 2 min.~~
-	~~#Added 350 μl Buffer N3 and inverted the tube immediately but gently 4–6 times. Solution became cloudy.~~
-	~~#Centrifuged for 10 min at 14,000 rpm. A compact white pellet formed.~~
-	~~#Applied the supernatants from step 4 to the QIAprep spin column by decanting.~~
-	~~#Centrifuged for 60 s. Discarded the flow-through.~~
-	~~#Washed the QIAprep spin column with 0.5 ml Buffer PB and centrifuged for 60 s. Discarded the flow-through.~~
-	~~#Washed QIAprep spin column by with 0.75 ml Buffer PE and centrifuged for 60 s.~~
-	~~#Placed the QIAprep column in a clean 1.5 ml microcentrifuge tube. Eluted with 30 μl water to the center of each QIAprep spin column, let stand for 2 min, and centrifuged for 60 s.~~
-	* digested promoter (R0010) and GFP (E0241) plasmids
-	** R0010 digested with SpeI and PstI in order to leave it attached at upstream end to the plasmid backbone (otherwise, the fragment would only be ~200 bp long, which is a little too short for electrophoresis with much longer fragments
-	** E0241 digested with XbaI and PstI in order to cleave it as a fragment
-	~~#The following ingredients were each added to four 1.5 ml centrifuge tubes:~~
-	~~#*10 μl water~~
-	~~#*8 μl DNA from the previous step (R0010-1, R0010-2, E0241-1, E0241-2 in respective tubes)~~
-	~~#*2.5 μl 10x NEB buffer (#2 for R0010-01 and R0010-2, #3 for E0241-1 and E0241-2)~~
-	~~#*2.5 μl 10x BSE~~
-	~~#*1.0 μl 1:1 diluted enzyme A (SpeI for R0010-1 and R0010-2, XbaI for E0241-1 and E0241-2)~~
-	~~#*1.0 μl 1:1 diluted enzyme B (PstI for all).~~
-	~~#Tubes were incubated at 37°C for 90 min.~~
-	~~#Tubes were incubated at 80°C for 15 min in order to inactivate the enzymes.~~
-	~~#All four samples were run on a 1% agarose gel:~~
-	~~{\| {{table}}~~
-	~~\| align="center" style="background:#f0f0f0;"\|'''Lane'''~~
-	~~\| align="center" style="background:#f0f0f0;"\|'''Contents'''~~
-	~~\| align="center" style="background:#f0f0f0;"\|'''Loading Buffer'''~~
-	\|-
-	~~\| 0\|\|1kb DNA ladder (10 {{ul}})\|\|10x loading dye (1.1 {{ul}})~~
-	\|-
-	~~\| 1\|\|E0241-1 (15 {{ul}})\|\|10x loading dye (2.5 {{ul}})~~
-	\|-
-	~~\| 2\|\|E0241-2 (15 {{ul}})\|\|10x loading dye (2.5 {{ul}})~~
-	\|-
-	~~\| 3\|\|R0010-1 (15 {{ul}})\|\|10x loading dye (2.5 {{ul}})~~
-	\|-
-	~~\| 4\|\|R0010-2 (15 {{ul}})\|\|10x loading dye (2.5 {{ul}})~~
-	\|}
-
-	~~====Tue Jun 13====~~
-
-	~~DNA nanostructure reaction PCR~~
-	* order of lanes: 1) 1kb ladder, 2) full rxn (oligos + scaffold), 3) just scaffold, 4) just oligos
-	* run on 2% agarose gel
-	* appears that reaction was successful: [[Media:Tc_mm_pt_lh_folding_0613.jpg\|gel image]] (leftmost four lanes) shows expected results
-	** assembled nanostructure runs slightly faster than scaffold, oligos appear as a smear of short DNAs
-
-	~~Transformation of standard components~~
-	* results of plated bacteria: all three plates showed colony growth, negative control did not
-	* colony selection and amplification
-	~~# Seven culture tubes were filled with 5 mL LB medium and 50 μl of 5 mg/mL ampicillin~~
-	~~# Colonies were selected from the three plates (three from R0010, two from E7104, two from E0241) and were transferred into respective culture tubes with a sterilized pick~~
-	~~# Culture tubes were incubated, with shaking, at 37°C for 18 h.~~
-	~~# Plates were stored at 4°C.~~
-
-	~~====Mon Jun 12====~~
-
-	~~DNA nanostructure reaction PCR~~
-	* [[Folding DNA nanostructures\|standard protocol]] with materials from Shawn
-
-	~~Transformation of standard components~~
-	* goal: insert three BioBrick plasmids (already containing BioBricks) into ''E. coli'' in order to amplify them
-	** a positive control (E7104) with the T7 promoter upstream of GFP
-	** the lac operon (R0010)
-	** GFP (E0241)
-	* transformation protocol:
-	~~# 1 {{ul}} each of E7104, R0010, E0241, and water into separate microcentrifuge tubes that each contain 30 {{ul}} of competent cells~~
-	~~# Incubated the tubes on ice for 20 min.~~
-	~~# Heat shocked the tubes at 42°C for 30 s.~~
-	~~# Added 200 {{ul}} SOC to each tube.~~
-	~~# Incubated, with shaking, at 37°C for 1 h.~~
-	~~# Plated the mixture on carbomycin plates and incubated at 37°C for 24 h.~~

	==Literature summaries and brainstorming==		==Literature summaries and brainstorming==

Matthewmeisel: /* Notebook */

2006-07-10T20:37:15Z

Notebook

←Older revision		Revision as of 20:37, 10 July 2006
Line 9:		Line 9:

	<calendar>		<calendar>
-	name=User:~~JeffreyLau~~/Notebook	+	name=User:Matthewmeisel/Notebook
	date=2006/07/01		date=2006/07/01
	view=threemonths		view=threemonths

Matthewmeisel: calendar

2006-07-10T20:36:54Z

calendar

←Older revision		Revision as of 20:36, 10 July 2006
Line 7:		Line 7:

	==Notebook==		==Notebook==
		+
		+	<calendar>
		+	name=User:JeffreyLau/Notebook
		+	date=2006/07/01
		+	view=threemonths
		+	format=%name/%year-%month-%day
		+	weekstart=14
		+	</calendar>

	===Week 2===		===Week 2===

Matthewmeisel: /* Working file scrapbook */

2006-06-28T00:34:53Z

Working file scrapbook

←Older revision		Revision as of 00:34, 28 June 2006
Line 2:		Line 2:

	==Working file scrapbook==		==Working file scrapbook==
		+	*[[Image:Igemharv06_msm3.2_-Converted-.ai]]
	*[[Image:Igemharv06_msm3.2.ai]]		*[[Image:Igemharv06_msm3.2.ai]]
	*[[Image:Igemharv06_msmrect.ai]]		*[[Image:Igemharv06_msmrect.ai]]

Matthewmeisel: /* Notebook */ scrapbook

2006-06-27T19:22:00Z

Notebook: scrapbook

←Older revision		Revision as of 19:22, 27 June 2006
Line 1:		Line 1:
	Quick link: [[iGEM:Harvard/2006]]		Quick link: [[iGEM:Harvard/2006]]

-	==~~Notebook~~==	+	==Working file scrapbook==
-	[[Image:Igemharv06_msmrect.ai]]	+	*[[Image:Igemharv06_msm3.2.ai]]
		+	*[[Image:Igemharv06_msmrect.ai]]

		+	==Notebook==

	===Week 2===		===Week 2===

Matthewmeisel: /* Notebook */

2006-06-26T21:15:33Z

Notebook

←Older revision		Revision as of 21:15, 26 June 2006
Line 2:		Line 2:

	==Notebook==		==Notebook==
		+	[[Image:Igemharv06_msmrect.ai]]
		+

	===Week 2===		===Week 2===

Matthewmeisel: tues digest

2006-06-20T19:37:36Z

tues digest

←Older revision		Revision as of 19:37, 20 June 2006
Line 23:		Line 23:
	#Washed QIAprep spin column by with 0.75 ml Buffer PE and centrifuged for 60 s.		#Washed QIAprep spin column by with 0.75 ml Buffer PE and centrifuged for 60 s.
	#Placed the QIAprep column in a clean 1.5 ml microcentrifuge tube. Eluted with 30 μl water to the center of each QIAprep spin column, let stand for 2 min, and centrifuged for 60 s.		#Placed the QIAprep column in a clean 1.5 ml microcentrifuge tube. Eluted with 30 μl water to the center of each QIAprep spin column, let stand for 2 min, and centrifuged for 60 s.
		+	* digested to verfiy that the insert was inserted
		+	#The following ingredients were each added to a 1.5 ml centrifuge tube:
		+	#*10 μl water
		+	#*8 μl DNA from the previous step
		+	#*2.5 μl 10x NEB buffer (#2)
		+	#*2.5 μl 10x BSE
		+	#*1.0 μl 1:1 diluted enzyme A (SpeI)
		+	#*1.0 μl 1:1 diluted enzyme B (XbaI).
		+	#Tubes were incubated at 37°C for 3 h.
		+	#Tubes were incubated at 80°C for 15 min in order to inactivate the enzymes.
		+	#10 {{ul}} of digest reaction and 10 {{ul}} water were mixed and run in a 1.2% agarose Invitrogen E-gel for 30 min. (ladder in lane 1, our sample in lane 4).

	====Mon Jun 19====		====Mon Jun 19====

Matthewmeisel at 15:10, 20 June 2006

2006-06-20T15:10:20Z

←Older revision		Revision as of 15:10, 20 June 2006
Line 50:		Line 50:
	# Added 200 {{ul}} SOC to each tube.		# Added 200 {{ul}} SOC to each tube.
	# Incubated, with shaking, at 37°C for 1 h.		# Incubated, with shaking, at 37°C for 1 h.
-	# Plated the mixture on ~~ampicillin~~ plates and incubated at 37°C for 24 h, then stored at 4°C.	+	# Plated the mixture on carbomycin plates and incubated at 37°C for 24 h, then stored at 4°C.

	====Wed Jun 14====		====Wed Jun 14====
Line 131:		Line 131:
	# Added 200 {{ul}} SOC to each tube.		# Added 200 {{ul}} SOC to each tube.
	# Incubated, with shaking, at 37°C for 1 h.		# Incubated, with shaking, at 37°C for 1 h.
-	# Plated the mixture on ~~ampicillin~~ plates and incubated at 37°C for 24 h.	+	# Plated the mixture on carbomycin plates and incubated at 37°C for 24 h.

	==Literature summaries and brainstorming==		==Literature summaries and brainstorming==