Team:Washington/Notebook

From 2009.igem.org

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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====
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[[Team:Washington/Notebook/Ni-column|Ni-column Set up]]
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#Transfer 2mL NiNTA beads (Quigen) to column
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#wash column with 10mL dH2O
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#Equillibrate column with 10mL running buffer (usually PBS)
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#To reuse column
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##Wash with 12mL dH2O
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##Wash with 12mL 100mM EDTA
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##Wash with 12mL dH2O
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##wash with 12mL 100mM Ni(SO4)
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##Wash with 12mL dH2O
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##Add 12mL 20% Ethanol run till ~5mL remains in column
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##cap for use later
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==== Supernatant Protein Purification, 2mL ====
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[[Team:Washington/Notebook/2mL_purification|Supernatant Protein Purification, 2mL]]
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#Inoculate 50mL culture of TB with ~750uL overnight culture
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#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
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#Bind Protein to column
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##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
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#Wash column
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##Apply 500uL PBS to column, aspirate to suspend beads
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##Spin column for 30sec at 500rpm, discard flow through
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##Repeat 1-2
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#Elute protein off of column
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##Make PBS with 1mg/mL BSA and 100uM imidazole
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##Add 200uL PBS + 1mg/mL BSA + 100uM imidazole to column aspirate to mix beads
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##Let sit for 2 min
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#Place Column in clean collection tube
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#Spin column for 5min at 500rpm
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#FLOW THROUGH IS YOUR PURIFIED PROTEIN
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==== Gene Synthesis ====
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[[Team:Washington/Notebook/gene_synthesis|Gene Synthesis]]
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#Generate Oligo's
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##Go to: http://helixweb.nih.gov/dnaworks/
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##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
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####Bio-Brick requires EcoRI, XbaI, SpeI, and PstI, others can be chosen if desired
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###Leave rest of options default
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##Enter Sequences
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###Click "Add Sequence Field" twice under Sequence formats
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###Imput your header sequence (select Nucleotide, this contains your cut sites,spacers, etc for subsequent cloning)
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####eg. GGATAGGA CATATG
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###Enter you protein sequence (select protein)
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###Imput your tail sequence (select Nucleotide, this contains your cut sites,spacers, etc for subsequent cloning)
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####e.g. CTCGAG ATTCGATG
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##RUN
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###If nothing is running make sure there are no blank new lines in your sequence section!
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##Choose your favorite oligo set to synthesize your gene
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###Usually look for the best scoring with the closest Tm's and oligo lengths
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##Design two additional oligos to amplify your gene
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###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.
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##ORDER
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###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.
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#Synthesize Gene
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##Dilute all oligos to 100uM
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##Mix together
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###add 5uL of each into a new master tube
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##Setup Synthesis PCR Reaction (have tried Taq, Vent, and PfuTurbo. Results are always best with Phusion)
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###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
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###0.5uL Phusion polymerase
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###36.5uL diH2O
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##Synthesis PCR Reaction
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###98C - 30s
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###98C - 10s
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###63C - 10s
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###72C - 30s/kb target gene
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###Repeat 2-4 29x
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###72C - 5min
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###10C - forever
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##Setup Amplification PCR Reaction
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###1uL FROM UNPURIFIED SYNTHESIS REACTION
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###1uL 25mM dNTP's
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###10uL Phusion HF Buffer
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###0.5uL Forward Primer (Tm 65)
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###0.5uL Reverse Primer (Tm 65)
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###0.5uL Phusion polymerase
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###36.5uL diH2O
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##Amplification PCR Reaction
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###98C - 30s
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###98C - 10s
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###63C - 10s
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###72C - 30s/kb target gene
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###Repeat 2-4 29x
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###72C - 5min
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###10C - forever
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#Run a 1% agarose gel of the synthesis and amplification reaction
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##5uL sample, 1uL loading buffer
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##You should see a smear from 60bp to over your gene length in the synthesis reaction
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##In the Amplification reaction a single band with your gene of interest should be there
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#Continue on with standard cloning!
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##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.
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#TROUBLESHOOTING
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##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.
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##First I often remove the annealing step use a 2 step protocol (Denature – Amplify x 29)!
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##Still, if no gene is amplified I run a gradient PCR
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##Then I try 0.5M Betaine (from 5M stock), or 5% DMSO
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##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.
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==== Colony PCR ====
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[[Team:Washington/Notebook/colony_PCR|Colony PCR]]
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#Prepare one sterile 0.6mL tube with the following reaction mixture for each colony you intend to pick.
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##5uL Qiagen Master Mix
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##1uL 40uM VF2
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##1uL 40uM VR
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#Prepare one sterile 0.6mL tube with 20uL sterile diH2O for each colony you intend to pick.
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#Pick colonies
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##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
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#Run reaction
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##94C - 3min
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##94C - 30s
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##55C - 30s
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##72C - 1min / kb gene
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##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 ====
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#Start with first 23 coding nucleotides of gene, eg. 5'-atgcgtaaaggagaagaacttt...-3'
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#Replace the atg start codon with the XbaI site: 5'-TCTAGA-3', eg. 5'-TCTAGAcgtaaaggagaagaacttt-3'
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#Add 6-8 random nucleotides to the 5' end of the primer, eg. 5'-cgggcTCTAGAcgtaaaggagaagaacttt-3'
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#Tweak the 3' end of the primer (add /remove nucleotides) so that the annealing temperature is close to that of VR
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#Amplify your gene using the designed forward oligo and VR
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#PCR purify the PCR product
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##To ensure that the proper size fragment was amplified 5uL of PCR reaction can be run on an agarose gel
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#Digest PCR product with XbaI and PstI
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##PCR purify
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#Digest Vector with NheI and PstI
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##PCR purify
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#Mix insert and vector in 3:1 ratio and ligate
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#Transform into competent cells
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#Screen cells for correct insert using VF2 and VR
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==== Gene Assembly using the NheI sites ====
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[[Team:Washington/Notebook/NheI_PstI|Gene Assembly using the NheI and PstI sites]]
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#Start with first 23 coding nucleotides of gene, eg. 5'-atgcgtaaaggagaagaacttt...-3'
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#Replace the atg start codon with the XbaI site: 5'-TCTAGA-3', eg. 5'-TCTAGAcgtaaaggagaagaacttt-3'
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#Add 6-8 random nucleotides to the 5' end of the primer, eg. 5'-cgggcTCTAGAcgtaaaggagaagaacttt-3'
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#Tweak the 3' end of the primer (add /remove nucleotides) so that the annealing temperature is close to 58C
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#Next start with the last 23 coding nucleotides eg.5'-tattttcagggtgctagctaa-3'
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#Remove the stop codon(s), in this case taa, and replace with the SpeI cut site ACTAGT, eg. 5'-tattttcagggtgctagcACTAGT-3'
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#Add 6-8 random nucleotides to the 3' end of the primer, eg. 5'-tattttcagggtgctagcACTAGTctgggtc-3'
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#REVERSE COMPLEMENT PRIMER, eg. 5'-tattttcagggtgctagcACTAGTctgggtc-3'  --> 5'-gacccagACTAGTgctagcaccctgaaaata-3'
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##Tweak the 3' end of the primer (add /remove nucleotides) so that the annealing temperature is close to 58C
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#Amplify your gene using the designed forward and reverse primers
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#PCR purify the PCR product
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##To ensure that the proper size fragment was amplified 5uL of PCR reaction can be run on an agarose gel
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#Digest PCR product with XbaI and SpeI
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##PCR Purify
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#Digest Vector with NheI and CIP
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##PCR purify
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#Mix insert and vector in 3:1 ratio and ligate
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#Transform into competent cells
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#SCREEN CELLS FOR CORRECT INSERT ORIENTATION by colony PCR using VF2 and custom reverse oligo
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[[Team:Washington/Notebook/NheI|Gene Assembly using the NheI site]]

Revision as of 04:17, 15 October 2009

Uw title logo.png

Protocols

Protein Gel

Microscope

Flow Cytometry

Supernatant Protein Purification, 50mL

Ni-column Set up

Supernatant Protein Purification, 2mL

Gene Synthesis

Colony PCR

Gene Assembly using the NheI and PstI sites

Gene Assembly using the NheI site


A ROUGH TIMELINE OF THE PROJECT!

Retrieved from "http://2009.igem.org/Team:Washington/Notebook"