Team:Washington/Notebook
From 2009.igem.org
(Difference between revisions)
Line 140: | Line 140: | ||
##ORDER | ##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. | ###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 | ||
+ | ##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 | ||
+ | ###1uL 25mM dNTP's | ||
+ | ###10uL Phusion HF Buffer | ||
+ | ###0.5uL Forward Oligo | ||
+ | ###0.5uL Reverse Oligo | ||
+ | ###0.5uL Phusion polymerase | ||
+ | ###36.5uL diH2O | ||
+ | ##Synthesis PCR Reaction | ||
+ | ###98C - 30s | ||
+ | ###98C - 10s | ||
+ | ###63C - 10s | ||
+ | ###72C - 30s/kb target gene | ||
+ | ###Repeat 2-4 29x | ||
+ | ###72C - 5min | ||
+ | ###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) | ||
+ | ###0.5uL Reverse Primer (Tm 65) | ||
+ | ###0.5uL Phusion polymerase | ||
+ | ###36.5uL diH2O | ||
+ | ##Amplification PCR Reaction | ||
+ | ###98C - 30s | ||
+ | ###98C - 10s | ||
+ | ###63C - 10s | ||
+ | ###72C - 30s/kb target gene | ||
+ | ###Repeat 2-4 29x | ||
+ | ###72C - 5min | ||
+ | ###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. | ||
Revision as of 02:37, 15 October 2009
A DETAILED DESCRIPTION OF THE PROTCOLS WE USED
- Gene Synthesis
- Colony PCR
- Assembly
- Cloning
- Expression
- Purification
Protein Gel
- Set up overnights of parts 48-51
- Dilute 1 ul overnight into 1ml
- Add 1 mm IPTG and let grow for four hours
- After cells have all grown up uniformly start boiling water for the boil step
- Add 100uL of overnight to a 1.5mL tube
- Pellet by spinning at max speed for 30 secs in the microcentrifuge
- discard supernatent
- Pull an aliquot of 5x sample loading buffer out of the freezer and thaw
- Add 20uL BME to aliquot
- Resuspend samples in 50uL sample loading buffer (pipette up/down)
- Boil samples for 10 minutes
- While boiling, prepare 500mL 1x SDS buffer:
- 50mL 10x buffer to 450mL water
- Take a gel out of the fridge and and put it in the gel box (keep the gel container for staining!!!)
- Pour the mixed buffer solution into the half of the gel box that the gel is in
- Remove the gel comb
- Fill the little container on the top of the gel until it's about 0.5 cm from the top with buffer
- Remove any bubbles in the wells
- Spin down samples for a few seconds
- Vortex samples
- Load 3uL into each well
- Load 10uL of ladder in appropriate wells
- Run at 180V until the dye is about to fall off the gel
Microscope
- Set up overnights of parts 48-51. Let grow overnight.
- Dilute 1 ul overnight into 1ml
- Add 1 mm IPTG and let grow for four hours
- After cells have grown up, place in flourophore (1 um) and allow time to bind (1 hour)
- Also place 1 ul beads in 1 ml along with 1 ul flourophore.
- Allow beads to bind to flourophore, then spin beads down, remove supernatent and replace with 1 ml water.
- Next place the cells and the beads under the microscope
Flow Cytometry
- Set up overnights of parts 48-51. Let grow overnight.
- Dilute 1 ul overnight into 1ml
- Add 1 mm IPTG and let grow for four hours
- After cells have grown up, place in flourophore (1 um) and allow time to bind (1 hour)
- Also place 1 ul beads in 1 ml along with 1 ul flourophore.
- Allow beads to bind to flourophore, then spin beads down, remove supernatent and replace with 1 ml water.
- Read the samples through the flow cytometer
Supernatant Protein Purification, 50mL
- Inoculate 50mL culture of TB with ~750uL overnight culture
- Grow a 37c until OD600: 0.4
- Inoculate cells with IPTG so that the final concentration is 0.5mM (25uL of 1M IPTG for 50mL culture)
- Grow cultures until OD600: 4, or use time points if looking for comparison in protein in supernatant
- Transfer culture to 50mL Falcon Tube
- Centrifuge at 8000rpm for 20 min to pellet cells
- Pour supernatant into 60mL syringe with 0.45uM filter attached
- Apply filtered supernatant to Ni-column (see Ni-column Set up for directions)
- Collect flow through and re-apply to column
- Wash column with 12mL PBS
- Elute column into 15mL condenser tube with 12mL PBS with 100mM imidazole
- Spin elutant in condenser tube until desired volume (~500uL) this is you purified protein.
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
- Wash with 12mL dH2O
- wash with 12mL 100mM Ni(SO4)
- Wash with 12mL dH2O
- Add 12mL 20% Ethanol run till ~5mL remains in column
- cap for use later
Supernatant Protein Purification, 2mL
- Inoculate 50mL culture of TB with ~750uL overnight culture
- Grow a 37c until OD600: 0.4
- Inoculate cells with IPTG so that the final concentration is 0.5mM (25uL of 1M IPTG for 50mL culture)
- Grow cultures until OD600: 4, or use time points if looking for comparison in protein in supernatant
- Prep NiNTA columns
- Place micro-centrifuge columns in collection tubes
- In micro-centrifuge columns add 200uL NiNTA beads
- Add 500uL PBS, aspirate to thoroughly rinse columns
- Spin columns with collection tubes for 30sec at 500rpm
- Transfer 2mL of growing culture to eppendorf tube
- Spin tube for 20min at 8000 rpm
- Remove supernatant, carefully as to not disrupt the pellet and set aside
- Bind Protein to column
- Add 500uL supernatant to the column
- Spin for 30sec at 500rpm
- Discard flow through
- Repeat 1-3 until all supernatant has run though the column
- Wash column
- Apply 500uL PBS to column, aspirate to suspend beads
- Spin column for 30sec at 500rpm, discard flow through
- Repeat 1-2
- 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
- Let sit for 2 min
- Place Column in clean collection tube
- Spin column for 5min at 500rpm
- FLOW THROUGH IS YOUR PURIFIED PROTEIN
Gene Synthesis
- Generate Oligo's
- Go to: http://helixweb.nih.gov/dnaworks/
- Set parameters
- Enter you job title and email
- Choose E.Coli Class II for codon frequency
- Set Annealing temperature to 60
- Maximize oligo length for cheapest oligo (60 for most companies)
- Set Number of solutions = 10
- Select Non-degenerate sites to avoid
- Bio-Brick requires EcoRI, XbaI, SpeI, and PstI, others can be chosen if desired
- 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)
- 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
- 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
- 1uL 25mM dNTP's
- 10uL Phusion HF Buffer
- 0.5uL Forward Oligo
- 0.5uL Reverse Oligo
- 0.5uL Phusion polymerase
- 36.5uL diH2O
- Synthesis PCR Reaction
- 98C - 30s
- 98C - 10s
- 63C - 10s
- 72C - 30s/kb target gene
- Repeat 2-4 29x
- 72C - 5min
- 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)
- 0.5uL Reverse Primer (Tm 65)
- 0.5uL Phusion polymerase
- 36.5uL diH2O
- Amplification PCR Reaction
- 98C - 30s
- 98C - 10s
- 63C - 10s
- 72C - 30s/kb target gene
- Repeat 2-4 29x
- 72C - 5min
- 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.
A ROUGH TIMELINE OF THE PROJECT!