http://2012.igem.org/wiki/index.php?title=Special:Contributions/Tjiang&feed=atom&limit=50&target=Tjiang&year=&month=2012.igem.org - User contributions [en]2024-03-28T10:31:54ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:Washington/Protocols/Gel_PurificationTeam:Washington/Protocols/Gel Purification2012-10-01T03:47:30Z<p>Tjiang: Created page with "=Gel Purification= #Using the QIAquick kit, first excise the DNA fragment form the agarose gel with a clean, sharp blade. #Weigh the gel slice in a tared colorless tube. Add 3 v..."</p>
<hr />
<div>=Gel Purification=<br />
<br />
#Using the QIAquick kit, first excise the DNA fragment form the agarose gel with a clean, sharp blade.<br />
#Weigh the gel slice in a tared colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100mg ~ 100µL). For >2% agarose gells, add 6 volumes Buffer QG.<br />
#Incubate at 50C for 10 min or until the gel has dissolved completely. Vortex every 2-3 min to help dissolve the gel.<br />
#After the gel has dissolved completely, make sure the mixture is yellow (similar to Buffer QG without the dissolved agarose). If the mixture is orange or violet, add 10µL 3M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.<br />
#Place a QIAquick spin column in a provided 2mL collection tube.<br />
#To bind DNA, apply sample to the column and centrifuge for 1 min.<br />
#Discard flow-through and place the column back into the same tube.<br />
#To wash, add 750µL Buffer PE to the column, let stand for 1 minute, and centrifuge for 1 min.<br />
#Discard flow-through and place the column back into the same tube.<br />
#Centrifuge the column in the tube again for 1 minute to remove residual wash buffer and discard the collection tube with the flow-through.<br />
#Place the column into a clean 1.5mL eppendorf tube.<br />
#To elute DNA, add 50uL Buffer EB to the center of the membrane in the column and centrifuge the column in the eppendorf tube for 1 minute. To get a higher DNA concentration, add 30µL Buffer EB to the center of the column membrane, let the column stand for 1min, and centrifuge in the eppendorf for 1 min.To further increase the concentration, letting the column stand for up to 4 minutes after the addition of the Buffer EB can help.<br />
#Nanodrop the DNA in the Buffer EB to get the concentration.</div>Tjianghttp://2012.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2012-10-01T03:29:54Z<p>Tjiang: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<h1 id=”General”>General Protocols</h1><br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/gel_electrophoresis General Agarose Gel Electrophoresis]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/PCR General PCR Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Digestion General Digestion Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Ligation General Ligation Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Transformation General Transformation Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Colony Colony PCR Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Competent Competent Cell Prep Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/expression_purification Standard 1L Expression Purification]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/gene_assembly Gene Assembly With Oligos]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/sequencing Sequencing]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/CompDesign Computational Protein Design]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Glycerol_Stocks Glycerol Stocks]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Gib_Rxn Gibson Cloning/Assembly]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Plas_DNA. Isolation of Plasmid DNA (miniprep)]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Overnights. Preparation of Overnight Cultures]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/PCR_Purification PCR Purification]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Gel_Purification Gel Purification]<br />
<br />
<h1 id=”Plastics”>Plastic Degradation</h1><br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/osmY_Assay osmY Assay]<br />
<br />
<h1 id=”Flu”>Flu Binders</h1><br />
[https://2012.igem.org/Team:Washington/Protocols/Kunkel Kunkel Mutagenesis]<br />
<br />
[http://www.bio.davidson.edu/Courses/molbio/kunkel/kunkel.html Overview of the Kunkel Mutagenesis process]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Display Yeast Surface Display]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Yeast Yeast Transformation]<br />
<br />
<h1 id=”Optogenetics”>Optogenetics</h1><br />
[https://2012.igem.org/Team:Washington/Protocols/Optogenetics/LightAppExperiments Light App Experiments]</div>Tjianghttp://2012.igem.org/Team:Washington/Protocols/PCR_PurificationTeam:Washington/Protocols/PCR Purification2012-10-01T03:26:35Z<p>Tjiang: Created page with "=PCR Purification= #Using the QIAquick kit, add 5 volumes of Buffer PB to 1 volume of the PCR sampe and mix. It is not necessary to remove mineral oil or kerosene. #If pH indica..."</p>
<hr />
<div>=PCR Purification=<br />
<br />
#Using the QIAquick kit, add 5 volumes of Buffer PB to 1 volume of the PCR sampe and mix. It is not necessary to remove mineral oil or kerosene.<br />
#If pH indicator I has been added to Buffer PB, check that the color of the mixture is yellow. If the mixture is orange or violet, add 10µL of 3M sodium acetate, pH 5.0 and mix. The color of the mixture will turn yellow.<br />
#Place a QIAquick spin column in a provided 2mL collection tube.<br />
#To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 minute.<br />
#Discard flow-through. Place the QIAquick column back into the same tube.<br />
#To wash, add 750µL Buffer PE to the column and centrifuge for 1 minute.<br />
#Discard flow-through and place the QIAquick column back in the same tube. Centrifuge the column for an additional 1 minute.<br />
#Discard collection tube and place the column in a clean 1.5mL eppendorf tube.<br />
#To elute DNA, add 50uL Buffer EB to the center of the membrane in the column and centrifuge the column in the eppendorf tube for 1 minute. To get a higher DNA concentration, add 30µL Buffer EB to the center of the column membrane, let the column stand for 1min, and centrifuge in the eppendorf for 1 min.<br />
#Nanodrop the DNA in the Buffer EB to get the concentration.</div>Tjianghttp://2012.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2012-10-01T03:16:06Z<p>Tjiang: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<h1 id=”General”>General Protocols</h1><br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/gel_electrophoresis General Agarose Gel Electrophoresis]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/PCR General PCR Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Digestion General Digestion Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Ligation General Ligation Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Transformation General Transformation Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Colony Colony PCR Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Competent Competent Cell Prep Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/expression_purification Standard 1L Expression Purification]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/gene_assembly Gene Assembly With Oligos]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/sequencing Sequencing]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/CompDesign Computational Protein Design]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Glycerol_Stocks Glycerol Stocks]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Gib_Rxn Gibson Cloning/Assembly]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Plas_DNA. Isolation of Plasmid DNA (miniprep)]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Overnights. Preparation of Overnight Cultures]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/PCR_Purification PCR Purification]<br />
<br />
<h1 id=”Plastics”>Plastic Degradation</h1><br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/osmY_Assay osmY Assay]<br />
<br />
<h1 id=”Flu”>Flu Binders</h1><br />
[https://2012.igem.org/Team:Washington/Protocols/Kunkel Kunkel Mutagenesis]<br />
<br />
[http://www.bio.davidson.edu/Courses/molbio/kunkel/kunkel.html Overview of the Kunkel Mutagenesis process]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Display Yeast Surface Display]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Yeast Yeast Transformation]<br />
<br />
<h1 id=”Optogenetics”>Optogenetics</h1><br />
[https://2012.igem.org/Team:Washington/Protocols/Optogenetics/LightAppExperiments Light App Experiments]</div>Tjianghttp://2012.igem.org/Team:Washington/Protocols/osmY_AssayTeam:Washington/Protocols/osmY Assay2012-10-01T03:07:23Z<p>Tjiang: </p>
<hr />
<div>=osmY Assay=<br />
<br />
#Make 500mL of M-9 media with 1x M9 salts, .1% glucose by mass, .5g casamino acids, and .001% biotin.<br />
#Make 4 liquid culture tubes each of sfGFP-osmY (n-c), osmY-GFP (Bright) (c-n), iGEM2011-003 sfGFP-mamI 1A3, iGEM2011-009 plac Bright 1A3, and no cells. All of these cultures are in 2mLs of M-9 media. Leave these tubes overnight.<br />
#The next day, take 700µL of media out of each culture tube. <br />
##Pipet 200µL of that 700 into a well in a clear, flat-bottom 96 well plate.<br />
##Pipet the other 500µL into a 1.5 mL eppendorf tube.<br />
#Centrifuge the eppendorf tubes containing the media for 3 minutes at 3g.<br />
#After spinning, remove the supernatent from each eppendorf tube and pipet it into a new eppendorf tube. Be sure not to disturb the cell pellets and keep the eppendorf tubes.<br />
#Take the new eppendorf tubes with supernatent only and centrifuge them for 10 minutes at 17g.<br />
#During this time, resuspend the cells in the old eppendorf tubes with 500µL of fresh M9-glucose media, the same media that the cells were grown in<br />
#After resuspending, take 200µL of the supernatant containing the resuspended cells and pipet it into the same 96 well plate the original culture was pipetted into.<br />
#Lastly, take the supernatent from the eppendorf tubes that have just been cenrifuged for 10 minutes and pipet 200µL from each tube into the 96 well plate. In total, 60 wells from the 96 well plate were used.</div>Tjianghttp://2012.igem.org/Team:Washington/Protocols/osmY_AssayTeam:Washington/Protocols/osmY Assay2012-10-01T03:04:08Z<p>Tjiang: Created page with "=osmY Assay= #Make M-9 media #Make 4 liquid culture tubes each of sfGFP-osmY (n-c), osmY-GFP (Bright) (c-n), iGEM2011-003 sfGFP-mamI 1A3, iGEM2011-009 plac Bright 1A3, and no ce..."</p>
<hr />
<div>=osmY Assay=<br />
<br />
#Make M-9 media<br />
#Make 4 liquid culture tubes each of sfGFP-osmY (n-c), osmY-GFP (Bright) (c-n), iGEM2011-003 sfGFP-mamI 1A3, iGEM2011-009 plac Bright 1A3, and no cells. All of these cultures are in 2mLs of M-9 media. Leave these tubes overnight.<br />
#The next day, take 700µL of media out of each culture tube. <br />
##Pipet 200µL of that 700 into a well in a clear, flat-bottom 96 well plate.<br />
##Pipet the other 500µL into a 1.5 mL eppendorf tube.<br />
#Centrifuge the eppendorf tubes containing the media for 3 minutes at 3g.<br />
#After spinning, remove the supernatent from each eppendorf tube and pipet it into a new eppendorf tube. Be sure not to disturb the cell pellets and keep the eppendorf tubes.<br />
#Take the new eppendorf tubes with supernatent only and centrifuge them for 10 minutes at 17g.<br />
#During this time, resuspend the cells in the old eppendorf tubes with 500µL of fresh M9-glucose media, the same media that the cells were grown in<br />
#After resuspending, take 200µL of the supernatant containing the resuspended cells and pipet it into the same 96 well plate the original culture was pipetted into.<br />
#Lastly, take the supernatent from the eppendorf tubes that have just been cenrifuged for 10 minutes and pipet 200µL from each tube into the 96 well plate. In total, 60 wells from the 96 well plate were used.</div>Tjianghttp://2012.igem.org/Team:Washington/Protocols/gene_assemblyTeam:Washington/Protocols/gene assembly2012-10-01T02:30:59Z<p>Tjiang: /* Gene Synthesis */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
<br />
=Gene Synthesis=<br />
#Generate Oligo's<br />
##Go to: http://helixweb.nih.gov/dnaworks/<br />
##Set parameters<br />
###Enter your job title and email<br />
###Choose E.Coli Class II for codon frequency<br />
###Set Annealing temperature to 60<br />
###Maximize oligo length for cheapest oligo (60 for most companies)<br />
###Set Number of solutions = 10<br />
###Select Non-degenerate sites to avoid<br />
####Bio-Brick requires EcoRI, XbaI, SpeI, and PstI, others can be chosen if desired<br />
###Leave rest of options default<br />
##Enter Sequences<br />
###Click "Add Sequence Field" twice under Sequence formats<br />
###Imput your header sequence (select Nucleotide, this contains your cut sites,spacers, etc for subsequent cloning)<br />
####eg. GGATAGGA CATATG<br />
###Enter you protein sequence (select protein)<br />
###Imput your tail sequence (select Nucleotide, this contains your cut sites,spacers, etc for subsequent cloning)<br />
####e.g. CTCGAG ATTCGATG<br />
##RUN<br />
###If nothing is running make sure there are no blank new lines in your sequence section!<br />
##Choose your favorite oligo set to synthesize your gene<br />
###Usually look for the best scoring with the closest Tm's and oligo lengths<br />
##Design two additional oligos to amplify your gene<br />
###A FORWARD and REVERSE oligo that complements your final DNA sequence with a Tm of 65. Just copy from the 5’ end of your first and last oligo from oligo’s reported from DNAWorks until you have a calculated Tm of 65 (20‐30bp, +/‐ 1deg). Try to make sure then ends are either G/C.<br />
##ORDER<br />
###I often try and order in plates (easier if ordering a lot) and make sure that the nmol of oligo is normalized. For IDT this is free, but that may differ for other companies.<br />
#Synthesize Gene<br />
##Dilute all oligos to 100uM<br />
###Resuspend oligos with water: uLs of water = concentration of oligos (in nm)*10<br />
##Mix together<br />
###add 5uL of each into a new master tube<br />
##Setup Synthesis PCR Reaction (have tried Taq, Vent, and PfuTurbo. Results are always best with Phusion)<br />
###1uL Oligo Mix<br />
###1uL 25mM dNTP's<br />
###10uL Phusion HF Buffer<br />
###0.5uL Forward Oligo<br />
###0.5uL Reverse Oligo<br />
###0.5uL Phusion polymerase<br />
###36.5uL diH2O<br />
##Synthesis PCR Reaction<br />
###98C - 30s<br />
###98C - 10s<br />
###63C - 10s<br />
###72C - 30s/kb target gene<br />
###Repeat 2-4 29x<br />
###72C - 5min<br />
###10C - forever<br />
##Setup Amplification PCR Reaction<br />
###1uL FROM UNPURIFIED SYNTHESIS REACTION<br />
###1uL 25mM dNTP's<br />
###10uL Phusion HF Buffer<br />
###0.5uL Forward Primer (Tm 65)<br />
###0.5uL Reverse Primer (Tm 65)<br />
###0.5uL Phusion polymerase<br />
###36.5uL diH2O<br />
##Amplification PCR Reaction<br />
###98C - 30s<br />
###98C - 10s<br />
###63C - 10s<br />
###72C - 30s/kb target gene<br />
###Repeat 2-4 29x<br />
###72C - 5min<br />
###10C - forever<br />
#Run a 1% agarose gel of the synthesis and amplification reaction<br />
##5uL sample, 1uL loading buffer<br />
##You should see a smear from 60bp to over your gene length in the synthesis reaction<br />
##In the Amplification reaction a single band with your gene of interest should be there<br />
#Continue on with standard cloning!<br />
##Make sure to sequence at least 4 clones. Often all 4 will be correct, but insertions,deletions, and spurious mutations sometime occur during the synthesis protocol.<br />
#TROUBLESHOOTING<br />
##Often I focus on the amplification step, assuming that there is a smear for the synthesis step on the gel and that smear covers the size of your gene of interest.<br />
##First I often remove the annealing step use a 2 step protocol (Denature – Amplify x 29)!<br />
##Still, if no gene is amplified I run a gradient PCR<br />
##Then I try 0.5M Betaine (from 5M stock), or 5% DMSO<br />
##Finally if nothing is working I break the gene into chunks and amplify smaller sections, then add those sections together and try to amplify the entire gene from the larger chunks.</div>Tjianghttp://2012.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2012-10-01T02:18:09Z<p>Tjiang: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<h1 id=”General”>General Protocols</h1><br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/gel_electrophoresis General Agarose Gel Electrophoresis]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/PCR General PCR Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Digestion General Digestion Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Ligation General Ligation Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Transformation General Transformation Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Colony Colony PCR Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Competent Competent Cell Prep Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/expression_purification Standard 1L Expression Purification]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/gene_assembly Gene Assembly With Oligos]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/sequencing Sequencing]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/CompDesign Computational Protein Design]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Glycerol_Stocks Glycerol Stocks]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Gib_Rxn Gibson Cloning/Assembly]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Plas_DNA. Isolation of Plasmid DNA (miniprep)]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Overnights. Preparation of Overnight Cultures]<br />
<br />
<h1 id=”Plastics”>Plastic Degradation</h1><br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/osmY_Assay osmY Assay]<br />
<br />
<h1 id=”Flu”>Flu Binders</h1><br />
[https://2012.igem.org/Team:Washington/Protocols/Kunkel Kunkel Mutagenesis]<br />
<br />
[http://www.bio.davidson.edu/Courses/molbio/kunkel/kunkel.html Overview of the Kunkel Mutagenesis process]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Display Yeast Surface Display]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Yeast Yeast Transformation]<br />
<br />
<h1 id=”Optogenetics”>Optogenetics</h1><br />
[https://2012.igem.org/Team:Washington/Protocols/Optogenetics/LightAppExperiments Light App Experiments]</div>Tjianghttp://2012.igem.org/Team:Washington/Protocols/Overnights.Team:Washington/Protocols/Overnights.2012-09-29T06:22:12Z<p>Tjiang: Created page with "{{Template:Team:Washington/Templates/Top}} =Preparation of Overnight Cultures= # Obtain a 14 mL Sterile Polypropylene Round-Bottom Tube. # Add ~ 3-5 mLs of an appropriate brot..."</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
<br />
=Preparation of Overnight Cultures=<br />
<br />
# Obtain a 14 mL Sterile Polypropylene Round-Bottom Tube.<br />
# Add ~ 3-5 mLs of an appropriate broth (either LB or TB)<br />
# Add an appropriate antibiotic if necessary and set this tube safely aside<br />
#* Amp (1:100)<br />
#* Chlor (1:1000)<br />
#* Kan (1:1000)<br />
# Prick the desired colony off a plate and suspend it in 50 uL PBS.<br />
#* Refer to the PBS stock making protocol!<br />
# Add 10 uL of the PBS sample and add it to the broth previously set aside.<br />
#* Incubate overnight cultures @ 37oC, overnight with shaking.</div>Tjianghttp://2012.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2012-09-29T06:21:29Z<p>Tjiang: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Protocols'''</big></big></big></big></center><br><br><br />
<br />
<h1 id=”General”>General Protocols</h1><br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/gel_electrophoresis General Agarose Gel Electrophoresis]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/PCR General PCR Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Digestion General Digestion Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Ligation General Ligation Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Transformation General Transformation Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Colony Colony PCR Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Competent Competent Cell Prep Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Kunkel Kunkel Mutagenesis]<br />
<br />
[http://www.bio.davidson.edu/Courses/molbio/kunkel/kunkel.html Overview of the Kunkel Mutagenesis process]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/expression_purification Standard 1L Expression Purification]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/gene_assembly Gene Assembly With Oligos]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/sequencing Sequencing]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/CompDesign Computational Protein Design]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Glycerol_Stocks Glycerol Stocks]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Gib_Rxn Gibson Cloning/Assembly]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Plas_DNA. Isolation of Plasmid DNA (miniprep)]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Overnights. Preparation of Overnight Cultures]<br />
<br />
<h1 id=”Plastics”>Plastic Degradation</h1><br />
[Protocols]<br />
<br />
<h1 id=”Optogenetics”>Optogenetics</h1><br />
[Protocols]<br />
<br />
<h1 id=”Flu”>Flu Binders</h1><br />
[Protocols]</div>Tjianghttp://2012.igem.org/Team:Washington/Protocols/Plas_DNA.Team:Washington/Protocols/Plas DNA.2012-09-29T06:14:46Z<p>Tjiang: Created page with "{{Template:Team:Washington/Templates/Top}} =Isolation of Plasmid DNA (miniprep)= # Gently vortex overnight culture(s) to mix cells. # Pipet ~1 mL of cells into a labeled micro..."</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
<br />
=Isolation of Plasmid DNA (miniprep)=<br />
<br />
# Gently vortex overnight culture(s) to mix cells.<br />
# Pipet ~1 mL of cells into a labeled microcentrifuge tube. Pellet the cells by centrifuging at 6000 X g. <br />
# Carefully pour off the supernatant without disturbing the pelleted cells.<br />
# Resuspend the pelleted cells in 250 μL of refrigerated buffer P1. <br />
# Add 250 μL of buffer P2 and thoroughly mix the tube by inverting 4-6 times. <br />
# Add 350 μL of buffer N3 and immediately (but gently) mix the tube by inverting 4-6 times. <br />
# Centrifuge the sample at ~17000 X g for 10 minutes. <br />
# Pour/Pipet the supernatant into a spin column (blue).<br />
# Centrifuge the sample for ~ 1 minute. Discard the flow-through. <br />
# Wash the sample by adding 500 μL buffer PB and centrifuge for ~ 1 minute. <br />
# Wash the sample by adding 750 μLbuffer PE and centrifuge for ~ 1 minute.<br />
# Discard the flow-through and centrifuge the sample for an additional 1 minute.<br />
# To Elute the DNA, place the spin column in a clean 1.5 μL (labeled) microcentrifuge. Add 30 μL of buffer EB and '''let Stand for 1 minute!''' <br />
# Centrifuge the sample for 1 minute.<br />
# Record the DNA concentration using the nanodrop program.<br />
#* Open the nanodrop program on the lab computer.<br />
#* Select "DNA"<br />
#* Once the program loads, pipet 1 ul of distilled H20 onto the machine. <br />
#* Hit "collect" in order to run the sample to clean the machine.Wipe off the remaining water with a kimwipe.<br />
#* Pipet 1 ul of EB buffer onto the machine. Hit "collect" in order to run the sample to clean the machine.Wipe off the remaining buffer with a kimwipe.<br />
#* Pipet 1 ul of a miniprep sample onto the machine. Hit "collect" in order to run the sample to clean the machine.Wipe off the remaining sample with a kimwipe, and record the concentration of the miniprep (ng/ul).<br />
#* Repeat the process of pipeting a sample and "collected" until all samples have been measured.</div>Tjianghttp://2012.igem.org/Team:Washington/ProtocolsTeam:Washington/Protocols2012-09-29T06:13:59Z<p>Tjiang: </p>
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__NOTOC__<br />
<br />
<center><big><big><big><big>'''Protocols'''</big></big></big></big></center><br><br><br />
<br />
<h1 id=”General”>General Protocols</h1><br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/gel_electrophoresis General Agarose Gel Electrophoresis]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/PCR General PCR Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Digestion General Digestion Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Ligation General Ligation Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Transformation General Transformation Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Colony Colony PCR Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Competent Competent Cell Prep Protocol]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Kunkel Kunkel Mutagenesis]<br />
<br />
[http://www.bio.davidson.edu/Courses/molbio/kunkel/kunkel.html Overview of the Kunkel Mutagenesis process]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/expression_purification Standard 1L Expression Purification]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/gene_assembly Gene Assembly With Oligos]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/sequencing Sequencing]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/CompDesign Computational Protein Design]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Glycerol_Stocks Glycerol Stocks]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Gib_Rxn Gibson Cloning/Assembly]<br />
<br />
[https://2012.igem.org/Team:Washington/Protocols/Plas_DNA. Isolation of Plasmid DNA (miniprep)]<br />
<br />
<h1 id=”Plastics”>Plastic Degradation</h1><br />
[Protocols]<br />
<br />
<h1 id=”Optogenetics”>Optogenetics</h1><br />
[Protocols]<br />
<br />
<h1 id=”Flu”>Flu Binders</h1><br />
[Protocols]</div>Tjianghttp://2012.igem.org/Team:Washington/PlasticsTeam:Washington/Plastics2012-09-19T21:57:04Z<p>Tjiang: </p>
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__NOTOC__<br />
<br />
<h1 id='Background'>Background</h1><br />
<br />
<p>Plastics play an integral role in the modern world. Due to their relatively low cost of production, they serve to promote the development of industry and lower the cost of consumer goods but perhaps the largest advantage to using plastics is their convenience. Even though plastics are attractive materials to use because of their durability, that same characteristic makes plastic slow to degrade. However, spite of these characteristics, plastics are often used in packaging and other products that are are commonly considered to be "disposable." For example, the test tubes and pipet tips used in lab. Regardless of efforts to promote recycling, an ever-increasing amount of plastic is making its way to the ocean where it is slowly degrading and releasing toxins that poison the wildlife. (http://coastalcare.org/2009/11/plastic-pollution/) </p><br />
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<h1 id='Methods'>Methods</h1><br />
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<h1 id='Results'>Results Summary</h1><br />
<h1 id='Future'>Future Directions</h1><br />
<h1 id='Parts'>Parts Submitted</h1></div>Tjianghttp://2012.igem.org/Team:Washington/PlasticsTeam:Washington/Plastics2012-09-19T21:55:30Z<p>Tjiang: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<h1 id='Background'>Background</h1><br />
<br />
<p>Plastics play an integral role in the modern world. Due to their relatively low cost of production, they serve to promote the development of industry and lower the cost of consumer goods but perhaps the largest advantage to using plastics is their convenience. Even though plastics are attractive materials to use because of their durability, that same characteristics makes plastics and slow to degrade. However, spite of these characteristics, plastics are often used in packaging and other products that are are commonly considered to be "disposable." For example, the test tubes and pipet tips used in lab. Regardless of efforts to promote recycling, an ever-increasing amount of plastic is making its way to the ocean where it is slowly degrading and releasing toxins that poison the wildlife. (http://coastalcare.org/2009/11/plastic-pollution/) </p><br />
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<h1 id='Methods'>Methods</h1><br />
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<h1 id='Results'>Results Summary</h1><br />
<h1 id='Future'>Future Directions</h1><br />
<h1 id='Parts'>Parts Submitted</h1></div>Tjiang