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-	{|align="justify"	+	{{WashUHeader2011}}
-	|You can write a background of your team here.  Give us a background of your team, the members, etc.  Or tell us more about something of your choosing.	+
-	|[[Image:WashU_logo.png|200px|right|frame]]	+
-	|-	+
-	|	+
-	''Tell us more about your project.  Give us background.  Use this is the abstract of your project.  Be descriptive but concise (1-2 paragraphs)''	+
-	|[[Image:WashU_team.png|right|frame|Your team picture]]	+
-	|-	+
-	|	+
-	|align="center"|[[Team:WashU | Team Example]]	+
-	|}	+
-		+
-	<!--- The Mission, Experiments --->	+
-		+
-	{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"	+
-	!align="center"|[[Team:WashU|Home]]	+
-	!align="center"|[[Team:WashU/Team|Team]]	+
-	!align="center"|[https://igem.org/Team.cgi?year=2011&team_name=WashU Official Team Profile]	+
-	!align="center"|[[Team:WashU/Project|Project]]	+
-	!align="center"|[[Team:WashU/Parts|Parts Submitted to the Registry]]	+
-	!align="center"|[[Team:WashU/Modeling|Modeling]]	+
-	!align="center"|[[Team:WashU/Notebook|Notebook]]	+
-	!align="center"|[[Team:WashU/Safety|Safety]]	+
-	!align="center"|[[Team:WashU/Attributions|Attributions]]	+
-	|}	+
	==Notebook==		==Notebook==
-	[[Team:WashU/Lab Notebook for May, 2011|Lab Notebook-May]]	+	[[File:Lab Notebook.jpg|300px|thumb|right|]]
-		+
-	[[Team:WashU/Lab Notebook for June, 2011|Lab Notebook-June]]	+
-		+
-	[[Team:WashU/Lab Notebook for July, 2011|Lab Notebook-July]]	+
-		+
-	You should make use of the calendar feature on the wiki and start a lab notebook.  This may be looked at by the judges to see how your work progressed throughout the summer.  It is a very useful organizational tool as well.	+
-		+
-	===July 1===	+
-	Master mix composition (6 wells worth)	+
-		+
-	**15.0uL 10x buffer	+
-	**7.5 uL dNTP	+
-	**99 uL dH20	+
-	**1.5 uL Accutaq LA DNA polyermerase	+
-		+
-	= 20.5 ul/well	+
-		+
-	**1.5 uL forward primer	+
-	**1.5 uL reverse primer	+
-		+
-	**10ng Kan DNA (1mL using our 2x diluted sample)	+
-	**1.5ul of Nat ligation product	+
-		+
-	2nd attempt at PCR on the Nat ligation product:	+
-	*Nat was not detected on a gel but may be because a very small amount of product was made.	+
-	*Well 1,2,3 are PCR mix with Nat primers and 1.5uL of Nat ligation product per well	+
-	*Well 4 is positive control with Kan primers and Kan DNA	+
-	*Well 5 just has PCR mix with the Nat primers.	+
-		+
-	===July 5===	+
-	Ran PCR to amplify 100x diluted sample of URA3	+
-	positive control contained mastermix, LEU2 primers and 100x diluted LEU2 DNA.	+
-	negative control contained mastermix, 100x diluted URA3	+
-	Note: this PCR reaction failed, including the positive control	+
-		+
-	PCR Protocol for Plasmids Review	+
-		+
-	**2.5uL 10x buffer	+
-	**1.25 uL dNTP	+
-	**16.5 uL dH20	+
-	**0.25 uL Accutaq LA DNA polyermerase	+
-		+
-	**1.5 uL forward primer	+
-	**1.5 uL reverse primer	+
-	**1.0 ul diluted DNA	+
-		+
-	===July 7===	+
-	Note: the 10x dilution of URA3 was the most effective, positive with LEU2 failed.	+
-	Reran PCR reaction to amplify different concentrations of URA3.	+
-	For this reaction we used a 2x, 5x, 10x, and 15x diluted samples of URA3 - ran each sample twice	+
-	2x: 1.5ul DNA, 1.5ul dH20	+
-	5x: 1ul DNA, 4ul dH20	+
-	10x: 1ul DNA, 9ul dH20	+
-	15x: 1ul DNA, 14ul dH20	+
-	positive control contained mastermix, LEU2 primers, and 20x diluted LEU2 DNA	+
-	1st negative control contained the mastermix and just LEU2 primers	+
-	2nd negative control negative contained the mastermix and just URA3 2x diluted DNA	+
-		+
-	PCR Protocol for Plasmids Review	+
-		+
-	**2.5uL 10x buffer	+
-	**1.25 uL dNTP	+
-	**16.5 uL dH20	+
-	**0.25 uL Accutaq LA DNA polyermerase	+
-		+
-	**1.5 uL forward primer	+
-	**1.5 uL reverse primer	+
-	**1.0 ul diluted DNA	+
-		+
-	Transformation:	+
-	*We are still waiting for Leu2 dropout medium to come in.	+
-	*In the meantime, we have designed an experiment to confirm that we have the pRS425 plasmid. We will cut the plasmid with specific restriction enzymes and run the digest on a gel to measure the resulting lengths.	+
-	**Of the restriction enzymes we have, we find that BamHI, XbaI, PstI, and XhoI cut the plasmid once, and EcoRI cuts the plasmid twice. We will use EcoRI and compare the lengths of the two fragments. We will also cut with BamHI and run that in a separate lane to confirm the overall plasmid length.	+
-		+
-	===July 8===	+
-	Transformation:	+
-	*We digested the DNA and then ran the results on a gel.	+
-	**Into a dilution microcentrifuge tube, we used 2 uL DNA (concentration 235.4 ng/uL) and 248 uL ddH20.	+
-	**BamHI tube: 2 uL diluted DNA, 2 uL NEBuffer, 2 uL diluted BSA (10x), 1 uL BamHI enzyme, 13 uL ddH20	+
-	**EcoRI tube: 2 uL diluted DNA, 2 ul SH buffer, 1 uL EcoRI enzyme, 15 uL reaction	+
-	**Incubated both tubes in a 37 C water bath for one hour for digestion	+
-	**Ran the solutions on a gel	+
-	***5 uL loading dye into three tubes: BamHI, EcoRI, control (uncut plasmid DNA)	+
-	***Lane 1: 5 uL ladder	+
-	***Lanes 2 and 3: 5 uL each BamHI	+
-	***Lanes 4 and 5: 5 uL each EcoRI	+
-	***Lane 6: 5 uL control	+
-	***Ran it for one hour at 132 volts	+
-		+
-	===July 11===	+
-	PCR group:	+
-	Reran five wells of PCR on URA3 - used 10x dilution URA3 DNA in our wells.	+
-	Positive control contained 2ul of 20x diluted LEU2, LEU2 primers, and mastermix.	+
-	Negative control contained 1ul 10x dilution URA3 and mastermix --> check for Plasmid DNA	+
-		+
-	PCR Protocol for Plasmids Review	+
-		+
-	**2.5uL 10x buffer	+
-	**1.25 uL dNTP	+
-	**16.5 uL dH20	+
-	**0.25 uL Accutaq LA DNA polyermerase	+
-	**20.5 ul/well	+
-		+
-	**1.5 uL forward primer	+
-	**1.5 uL reverse primer	+
-	**1.0 ul diluted DNA	+
-		+
-	Transformation:	+
-	*Set up yeast overnight for BC178	+
-	*Ran gel again with lower concentration enzyme; it did not work again	+
-	*E. coli transformation of Leu2 and Ura3 and plated it with Leu2-deficient drop-out media we made	+
-		+
-	===July 12===	+
-	Transformation:	+
-	*Plates from yesterday	+
-	*Yeast transformation for BC178; we accidentally added 1 M LiAC instead of 100 uM, so we might have to redo it	+
-	*Set up E. coli overnight	+
-		+
-	===July 13===	+
-	PCR:	+
-		+
-	Colony PCR:	+
-		+
-	Protocol:	+
-		+
-	**Transfer a yeast colony to a solution of 0.2% SDS. The SDS page was made by adding 0.02g SDS to 10mL of water.	+
-	**Vortex for 15 sec	+
-	**Heat in PCR machine for 4 min at 90 degrees Celsius.2	+
-	**Microcentrifuge for 1 min.	+
-	**Pipet out the supernatent and store at -20 degrees Celsius.	+
-		+
-	PCR mix for 50uL reaction:	+
-		+
-	**5uL 10x PCR buffer	+
-	**1.5 uL 50mM MgCl2 (we used 3uL because we 25mM MgCl2)	+
-	**1uL of 10mM dNTPs	+
-	**2uL of 25% TritonX-100	+
-	**0.3 uL Taq polymersase	+
-	**31.7 uL water	+
-		+
-	**Add 43 uL of PCR mix to each PCR tube	+
-	**Then add 3 uL of both the forward and reverse primers	+
-	**Add 1uL of the DNA from yeast	+
-		+
-		+
-	We made 2 samples with Leu2 primers, 2 samples with Uras3 primers, a positive control with Uras3 primers and DNA, and a negative control with just yeast DNA.	+
-	Strain 827 was used	+
-		+
-	===July 14===	+
-	PCR:	+
-		+
-	Colony PCR:	+
-		+
-	Protocol:	+
-		+
-	**Transfer a yeast colony to a solution of 0.2% SDS. The SDS page was made by adding 0.02g SDS to 10mL of water.	+
-	**Vortex for 15 sec	+
-	**Heat in PCR machine for 4 min at 90 degrees Celsius.2	+
-	**Microcentrifuge for 1 min.	+
-	**Pipet out the supernatent and store at -20 degrees Celsius.	+
-		+
-	PCR mix for 50uL reaction:	+
-		+
-	**5uL 10x PCR buffer	+
-	**1.5 uL 50mM MgCl2 (we used 3uL because we 25mM MgCl2)	+
-	**1uL of 10mM dNTPs	+
-	**2uL of 25% TritonX-100. The 25% TritonX-100 was made by adding 6ul of TritonX-100 to 18ul of H20	+
-	**0.3 uL Taq polymersase	+
-	**31.7 uL water	+
-		+
-	**Add 43 uL of PCR mix to each PCR tube	+
-	**Then add 3 uL of both the forward and reverse primers	+
-	**Add 1uL of the DNA from yeast	+
-		+
-	We made 1 samples with Leu2 primers, 2 samples with Uras3 primers and a positive control with Uras3 primers and DNA	+	[[Team:WashU/Notebook/May2011|Lab Notebook-May]]
-	Strain 825 was used	+
-	===July 15===	+	[[Team:WashU/Notebook/June2011|Lab Notebook-June]]
-	Ordered Biobrick primers and all 4 cassette primers without homology in order to run colony PCR	+
-	===July 18===	+	[[Team:WashU/Notebook/July2011|Lab Notebook-July]]
-	Ran a PCR for the genes: CrtI, CrtE, CcD1, CcYB --> ran 3 PCR tubes for each gene	+
-	**1 positive control: Nat Genes + primers	+
-	**4 negative controls, 1 corresponding to each gene, contains only the mastermix soln and F/R primers	+
-	Recipe per sample for running plasmid DNA:	+	[[Team:WashU/Notebook/August2011|Lab Notebook-August]]
-	*2.5uL 10x buffer	+
-	*1.25 uL dNTP	+
-	*1.5 uL forward primer	+
-	*1.5 uL reverse primer	+
-	*16.5 uL dH20	+
-	*0.25 uL Accutaq LA DNA polyermerase	+
-	Transformation:	+	[[Team:WashU/Notebook/September2011|Lab Notebook-September]]
-	*We had made colonies of BC178, but we had gotten contamination in them, so we made new plates, this time with ampicillin, in hopes of getting rid of any E. coli that may have been in our test tubes. Also, we filter-sterilized our lithium acetate (just in case) and our PEG 3350.	+
-	*We set up new overnights to start over.	+

---

Latest revision as of 04:02, 28 September 2011

Notebook

Lab Notebook-May

Lab Notebook-June

Lab Notebook-July

Lab Notebook-August

Lab Notebook-September