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| {{Template:Team:Washington/Templates/Header}} | | {{Template:Team:Washington/Templates/Header}} |
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- | ==== Protocols ==== | + | = '''Protocols''' = |
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| [[Team:Washington/Notebook/protein_gel|Protein Gel]] | | [[Team:Washington/Notebook/protein_gel|Protein Gel]] |
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- | [[Team:Washington/Notebook/Microscope|Microscope]] | + | [[Team:Washington/Notebook/Microscope|Microscopy]] |
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| [[Team:Washington/Notebook/Flow_Cytometry|Flow Cytometry]] | | [[Team:Washington/Notebook/Flow_Cytometry|Flow Cytometry]] |
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| [[Team:Washington/Notebook/50mL_purification|Supernatant Protein Purification, 50mL]] | | [[Team:Washington/Notebook/50mL_purification|Supernatant Protein Purification, 50mL]] |
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- | ====Ni-column Set up====
| + | [[Team:Washington/Notebook/Ni-column|Ni-column Set up]] |
- | #Transfer 2mL NiNTA beads (Quigen) to column
| + | |
- | #wash column with 10mL dH2O
| + | |
- | #Equillibrate column with 10mL running buffer (usually PBS)
| + | |
- | #To reuse column
| + | |
- | ##Wash with 12mL dH2O
| + | |
- | ##Wash with 12mL 100mM EDTA
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- | ##Wash with 12mL dH2O
| + | |
- | ##wash with 12mL 100mM Ni(SO4)
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- | ##Wash with 12mL dH2O
| + | |
- | ##Add 12mL 20% Ethanol run till ~5mL remains in column
| + | |
- | ##cap for use later
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| | | |
- | ==== Supernatant Protein Purification, 2mL ====
| + | [[Team:Washington/Notebook/2mL_purification|Supernatant Protein Purification, 2mL]] |
- | #Inoculate 50mL culture of TB with ~750uL overnight culture
| + | |
- | #Grow a 37c until OD600: 0.4
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- | #Inoculate cells with IPTG so that the final concentration is 0.5mM (25uL of 1M IPTG for 50mL culture)
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- | #Grow cultures until OD600: 4, or use time points if looking for comparison in protein in supernatant
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- | #Prep NiNTA columns
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- | ##Place micro-centrifuge columns in collection tubes
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- | ##In micro-centrifuge columns add 200uL NiNTA beads
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- | ##Add 500uL PBS, aspirate to thoroughly rinse columns
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- | ##Spin columns with collection tubes for 30sec at 500rpm
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- | #Transfer 2mL of growing culture to eppendorf tube
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- | ##Spin tube for 20min at 8000 rpm
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- | ##Remove supernatant, carefully as to not disrupt the pellet and set aside
| + | |
- | #Bind Protein to column
| + | |
- | ##Add 500uL supernatant to the column
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- | ##Spin for 30sec at 500rpm
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- | ##Discard flow through
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- | ##Repeat 1-3 until all supernatant has run though the column
| + | |
- | #Wash column
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- | ##Apply 500uL PBS to column, aspirate to suspend beads
| + | |
- | ##Spin column for 30sec at 500rpm, discard flow through
| + | |
- | ##Repeat 1-2
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- | #Elute protein off of column
| + | |
- | ##Make PBS with 1mg/mL BSA and 100uM imidazole
| + | |
- | ##Add 200uL PBS + 1mg/mL BSA + 100uM imidazole to column aspirate to mix beads
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- | ##Let sit for 2 min
| + | |
- | #Place Column in clean collection tube
| + | |
- | #Spin column for 5min at 500rpm
| + | |
- | #FLOW THROUGH IS YOUR PURIFIED PROTEIN
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| | | |
- | ==== Gene Synthesis ====
| + | [[Team:Washington/Notebook/gene_synthesis|Gene Synthesis]] |
- | #Generate Oligo's
| + | |
- | ##Go to: http://helixweb.nih.gov/dnaworks/
| + | |
- | ##Set parameters
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- | ###Enter you job title and email
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- | ###Choose E.Coli Class II for codon frequency
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- | ###Set Annealing temperature to 60
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- | ###Maximize oligo length for cheapest oligo (60 for most companies)
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- | ###Set Number of solutions = 10
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- | ###Select Non-degenerate sites to avoid
| + | |
- | ####Bio-Brick requires EcoRI, XbaI, SpeI, and PstI, others can be chosen if desired
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- | ###Leave rest of options default
| + | |
- | ##Enter Sequences
| + | |
- | ###Click "Add Sequence Field" twice under Sequence formats
| + | |
- | ###Imput your header sequence (select Nucleotide, this contains your cut sites,spacers, etc for subsequent cloning)
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- | ####eg. GGATAGGA CATATG
| + | |
- | ###Enter you protein sequence (select protein)
| + | |
- | ###Imput your tail sequence (select Nucleotide, this contains your cut sites,spacers, etc for subsequent cloning)
| + | |
- | ####e.g. CTCGAG ATTCGATG
| + | |
- | ##RUN
| + | |
- | ###If nothing is running make sure there are no blank new lines in your sequence section!
| + | |
- | ##Choose your favorite oligo set to synthesize your gene
| + | |
- | ###Usually look for the best scoring with the closest Tm's and oligo lengths
| + | |
- | ##Design two additional oligos to amplify your gene
| + | |
- | ###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.
| + | |
- | ##ORDER
| + | |
- | ###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.
| + | |
- | #Synthesize Gene
| + | |
- | ##Dilute all oligos to 100uM
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- | ##Mix together
| + | |
- | ###add 5uL of each into a new master tube
| + | |
- | ##Setup Synthesis PCR Reaction (have tried Taq, Vent, and PfuTurbo. Results are always best with Phusion)
| + | |
- | ###1uL Oligo Mix
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- | ###1uL 25mM dNTP's
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- | ###10uL Phusion HF Buffer
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- | ###0.5uL Forward Oligo
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- | ###0.5uL Reverse Oligo
| + | |
- | ###0.5uL Phusion polymerase
| + | |
- | ###36.5uL diH2O
| + | |
- | ##Synthesis PCR Reaction
| + | |
- | ###98C - 30s
| + | |
- | ###98C - 10s
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- | ###63C - 10s
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- | ###72C - 30s/kb target gene
| + | |
- | ###Repeat 2-4 29x
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- | ###72C - 5min
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- | ###10C - forever
| + | |
- | ##Setup Amplification PCR Reaction
| + | |
- | ###1uL FROM UNPURIFIED SYNTHESIS REACTION
| + | |
- | ###1uL 25mM dNTP's
| + | |
- | ###10uL Phusion HF Buffer
| + | |
- | ###0.5uL Forward Primer (Tm 65)
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- | ###0.5uL Reverse Primer (Tm 65)
| + | |
- | ###0.5uL Phusion polymerase
| + | |
- | ###36.5uL diH2O
| + | |
- | ##Amplification PCR Reaction
| + | |
- | ###98C - 30s
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- | ###98C - 10s
| + | |
- | ###63C - 10s
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- | ###72C - 30s/kb target gene
| + | |
- | ###Repeat 2-4 29x
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- | ###72C - 5min
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- | ###10C - forever
| + | |
- | #Run a 1% agarose gel of the synthesis and amplification reaction
| + | |
- | ##5uL sample, 1uL loading buffer
| + | |
- | ##You should see a smear from 60bp to over your gene length in the synthesis reaction
| + | |
- | ##In the Amplification reaction a single band with your gene of interest should be there
| + | |
- | #Continue on with standard cloning!
| + | |
- | ##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.
| + | |
- | #TROUBLESHOOTING
| + | |
- | ##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.
| + | |
- | ##First I often remove the annealing step use a 2 step protocol (Denature – Amplify x 29)!
| + | |
- | ##Still, if no gene is amplified I run a gradient PCR
| + | |
- | ##Then I try 0.5M Betaine (from 5M stock), or 5% DMSO
| + | |
- | ##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.
| + | |
| | | |
- | ==== Colony PCR ====
| + | [[Team:Washington/Notebook/colony_PCR|Colony PCR]] |
- | #Prepare one sterile 0.6mL tube with the following reaction mixture for each colony you intend to pick.
| + | |
- | ##5uL Qiagen Master Mix
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- | ##1uL 40uM VF2
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- | ##1uL 40uM VR
| + | |
- | #Prepare one sterile 0.6mL tube with 20uL sterile diH2O for each colony you intend to pick.
| + | |
- | #Pick colonies
| + | |
- | ##Pick a single colony using a micropipettor with sterile tip. The pippettor should be set to 3uL
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- | ##Aspirate colony into 20uL diH2O vigorously to transfer cells to diH2O
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- | ##Transfer 3uL of diH2O containing cells to reaction mixture set up in step 1
| + | |
- | #Run reaction
| + | |
- | ##94C - 3min
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- | ##94C - 30s
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- | ##55C - 30s
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- | ##72C - 1min / kb gene
| + | |
- | ##repeat 2 - 4 29x
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- | ##72C - 10min
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- | ##4C - forever
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- |
| + | |
- | ==== Gene Assembly using the NheI and PstI sites ====
| + | |
- | #Start with first 23 coding nucleotides of gene, eg. 5'-atgcgtaaaggagaagaacttt...-3'
| + | |
- | #Replace the atg start codon with the XbaI site: 5'-TCTAGA-3', eg. 5'-TCTAGAcgtaaaggagaagaacttt-3'
| + | |
- | #Add 6-8 random nucleotides to the 5' end of the primer, eg. 5'-cgggcTCTAGAcgtaaaggagaagaacttt-3'
| + | |
- | #Tweak the 3' end of the primer (add /remove nucleotides) so that the annealing temperature is close to that of VR
| + | |
- | #Amplify your gene using the designed forward oligo and VR
| + | |
- | #PCR purify the PCR product
| + | |
- | ##To ensure that the proper size fragment was amplified 5uL of PCR reaction can be run on an agarose gel
| + | |
- | #Digest PCR product with XbaI and PstI
| + | |
- | ##PCR purify
| + | |
- | #Digest Vector with NheI and PstI
| + | |
- | ##PCR purify
| + | |
- | #Mix insert and vector in 3:1 ratio and ligate
| + | |
- | #Transform into competent cells
| + | |
- | #Screen cells for correct insert using VF2 and VR
| + | |
| | | |
- | ==== Gene Assembly using the NheI sites ====
| + | [[Team:Washington/Notebook/NheI_PstI|BioBrick Assembly using the NheI and PstI sites]] |
- | #Start with first 23 coding nucleotides of gene, eg. 5'-atgcgtaaaggagaagaacttt...-3'
| + | |
- | #Replace the atg start codon with the XbaI site: 5'-TCTAGA-3', eg. 5'-TCTAGAcgtaaaggagaagaacttt-3'
| + | |
- | #Add 6-8 random nucleotides to the 5' end of the primer, eg. 5'-cgggcTCTAGAcgtaaaggagaagaacttt-3'
| + | |
- | #Tweak the 3' end of the primer (add /remove nucleotides) so that the annealing temperature is close to 58C
| + | |
- | #Next start with the last 23 coding nucleotides eg.5'-tattttcagggtgctagctaa-3'
| + | |
- | #Remove the stop codon(s), in this case taa, and replace with the SpeI cut site ACTAGT, eg. 5'-tattttcagggtgctagcACTAGT-3'
| + | |
- | #Add 6-8 random nucleotides to the 3' end of the primer, eg. 5'-tattttcagggtgctagcACTAGTctgggtc-3'
| + | |
- | #REVERSE COMPLEMENT PRIMER, eg. 5'-tattttcagggtgctagcACTAGTctgggtc-3' --> 5'-gacccagACTAGTgctagcaccctgaaaata-3'
| + | |
- | ##Tweak the 3' end of the primer (add /remove nucleotides) so that the annealing temperature is close to 58C
| + | |
- | #Amplify your gene using the designed forward and reverse primers
| + | |
- | #PCR purify the PCR product
| + | |
- | ##To ensure that the proper size fragment was amplified 5uL of PCR reaction can be run on an agarose gel
| + | |
- | #Digest PCR product with XbaI and SpeI
| + | |
- | ##PCR Purify
| + | |
- | #Digest Vector with NheI and CIP
| + | |
- | ##PCR purify
| + | |
- | #Mix insert and vector in 3:1 ratio and ligate
| + | |
- | #Transform into competent cells
| + | |
- | #SCREEN CELLS FOR CORRECT INSERT ORIENTATION by colony PCR using VF2 and custom reverse oligo
| + | |
| | | |
| + | [[Team:Washington/Notebook/NheI|BioBrick Assembly using the NheI site]] |
| | | |
| + | [[Team:Washington/Notebook/SOEingPCR|SOEing PCR]] |
| + | |
| + | [[Team:Washington/Notebook/IMAC_protocol|Traditional Protein Purification (IMAC)]] |
| + | |
| + | [[Team:Washington/Notebook/Standard_curve|Generating a Standard curve for GFP concentration]] |
| + | |
| + | <br> |
| + | |
| + | = '''Project Time Line''' = |
| + | |
| + | |
| + | |
| + | *Winter Quarter |
| + | **Introduction to iGEM |
| + | **Synthetic Biology Seminar |
| + | **Plan for project ideas |
| + | |
| + | |
| + | *Spring Quarter |
| + | **Narrow down potential projects |
| + | **Choose Project |
| + | **Order oligos and start synthesizing genes |
| + | **Obtained Funding |
| + | **Stock Lab |
| + | |
| + | |
| + | *June |
| + | **Sequence genes |
| + | **Preliminary binding assays for biotinylated fluorophore |
| + | **Introduction to Fold-it as a tool for protein design |
| + | **Test target proteins for solubility and expression |
| + | **Start assembly of secretion genes |
| + | |
| + | |
| + | *July |
| + | **Develop and perform assays for testing legacy surface display bio-bricks |
| + | **Transfer prtDEF contig from secretion system into low copy plasmid |
| + | **Test target proteins for functionality |
| + | |
| + | |
| + | *August |
| + | **Finish assembly of secretion system |
| + | **Start cell lines containing various forms of secretion system, make competent |
| + | **Transform competent cells containing secretion system with target vector |
| + | **Test for secretion of target protein |
| + | **Start design of new display construct |
| + | |
| + | |
| + | *September |
| + | **Switch secretion system into new cell line, make cells competent |
| + | **Transform with target vector |
| + | **Test for secretion |
| + | **Start Presentation |
| + | **Start t-shirt design |
| + | **Insert streptavidin into new display vector |
| + | |
| + | |
| + | *October |
| + | **Fine tune secretion assay, adjust controls |
| + | **Finalize characterization of legacy parts |
| + | **Practice presentation |
| + | **Characterize target bio-bricks |
| + | **Prepare for Jamboree |
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- | A ROUGH TIMELINE OF THE PROJECT!
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| {{Template:Team:Washington/Templates/Footer}} | | {{Template:Team:Washington/Templates/Footer}} |