Team:Pittsburgh/Experiments
Pittsburgh iGEM 2019 Experimental Protocols
TABLE OF CONTENTS
Storage Buffer (Protein Purification)
Resuspension Buffer (Protein Purification)
Lysis Buffer (Protein Purification)
Wash Buffer (Protein Purification)
Elution Buffer (Protein Purification)
Dialysis Buffer (Protein Purification)
Splicing Buffer (Intein Assays)
Mootz Splicing Buffer (Intein Assays)
Coomassie Stain for Protein Gels
Denaturing SDS Sample Buffer (3x)
Transformation Storage Buffer (TSB)
Preparation of Competent Cells
Plasmid Screening (Colony PCR)
Microscope preparation (basic compound microscope)
Expression Optimization Protocol
Purification Optimization Protocol
Diafiltration -Buffer Exchange
Purified and Dialyzed Protein Assays
Buffers & Reagents
Antibiotic Concentrations
Volume of antibiotic to add in microliters per | ||||
Antibiotic | Stock Solution Name | Stock Concentration (mg/mL) | LB plates (20mL/plate) | LB Broth (1 mL) |
Kanamycin | Kan 30 | 30 | 33.33 | 1 |
Chloramphenicol | Chlor 30 | 30 | 20 | 0.67 |
Streptomycin | Strep 50 | 50 | 12 | 0.60 |
Ampicillin | Amp 100 | 100 | 20 | 0.50 |
Protein Buffers
Storage Buffer (Protein Purification)
Total volume 4 L
- 150 mM NaCl
- 20 mM TrisHCl pH 7.4
- 20% Glycerol
- 1 mM β-mercaptoethanol (Add right before using)
Resuspension Buffer (Protein Purification)
Total volume 1 L
- 50 mM HEPES, pH 8.0
- 150 mM NaCl
Lysis Buffer (Protein Purification)
100 mL, store at 4 ̊C
- 150 mM NaCl
- 20 mM Tris Base, pH 7.4
- 20 mM imidazole, pH 7.0
- 1 mM β-mercaptoethanol
- Add right before using
- 1 EDTA-free protease inhibitor tablets
- Add right before using
- 100 U DNase
- Add right before using
- 0.1% Triton-X 100
- Add right before using
Wash Buffer (Protein Purification)
Total volume 250 mL
- 150 mM NaCl
- 20 mM Tris Base, pH 7.4
- 40 mM imidazole, pH 7.0
- 1 mM β-mercaptoethanol
- Add right before using
Elution Buffer (Protein Purification)
Total volume 250 mL
- 150 mM NaCl
- 20 mM Tris Base, pH 7.4
- 200 mM imidazole, pH 7.0
- 1 mM β-mercaptoethanol
- Add right before using
Dialysis Buffer (Protein Purification)
Total volume 1 L
- 150 mM NaCl
- 20 mM Tris Base, pH 7.4
- 1 mM β-mercaptoethanol
- add right before using
Splicing Buffer (Intein Assays)
Total volume 250 mL
- 150 mM 5 M NaCl
- 20 mM 2 M Tris Base, pH 7.4
- 1 mM β-mercaptoethanol
- Add right before using
- Protease inhibitor tablet
- Add right before using
- 10% (v/v) Glycerol
Mootz Splicing Buffer (Intein Assays)
Stored in aliquots at -80°C
- 50 mM Tris-HCl at pH 7.0
- 300 mM NaCl
- 1 mM EDTA
- 10% (v/v) glycerol
- 2 mM DTT
1x SDS Running Buffer
Total volume 1L
- 14.4 g Glycine
- 3.0 g Tris Base
- 1 g SDS
5X ISO Buffer
Store at -20°C in 320 µL aliquots
- 3 mL of 1M Tris Base at pH 7.5
- 150 µL of 2 M MgCl2
- 240 µL of 100 mM dNTP mix
- 300 µL of 1 M DTT
- 1.5 g PEG-8000
- 600 µL of 50 mM NAD+
- Fill to 6 mL with deionized water
Coomassie Stain for Protein Gels
1 L, store in the dark at room temperature
- 60-80 mg Coomassie G-250
- 1 L MilliQ water
- Stir for several hours to dissolve
5X KCM
30 mL total. Sterile filter, store at 4°C
- 0.5 M KCl
- 0.15 M CaCl2
- 0.25 M MgCl2
Denaturing SDS Sample Buffer (3x)
10 mL total. Store at -20°C
- 1.5 mg Bromophenol Blue
- 0.60 g SDS
- 3.0 mL Glycerol
- 3.9 mL of 0.5 M Tris Base at pH 6.8
- 1.5 mL of β-mercaptoethanol
Transformation Storage Buffer (TSB)
50 mL total. Sterile filter and store at 4°C
- 20.5 mL LB broth at pH 6.5
- 10% PEG-3350
- 5% DMSO
- 10 mM MgCl2
- 10 mM MgSO2
Preparation of Competent Cells
Day 1:
- Grow a 5 mL LB culture from a freezer stock of the strain of cells at 37°C overnight
Day 2:
- Innoculate 100 mL LB media with 1mL of the culture from the day before
- Shake at 37°C to OD600=0.4-0.6
- Place culture on ice for 5 min
- Pellet cells at 4000 xg for 10 minutes
- Resuspend cells in 10 mL of chilled TSB
- Incubate on ice for 5 minutes
- Subpackage ino tubes (100 µL aliquots) and freeze in liquid nitrogen
- Store at -80°C
Cloning
Phusion Polymerase
PCR Component | Number of PCR Reactions* | ||||||
1 | 5 | 9 | 11 | 15 | 16 | 21 | |
5X GC Buffer | 10 | 50 | 90 | 110 | 150 | 160 | 210 |
dNTPs (10mM) | 1 | 5 | 9 | 11 | 15 | 16 | 21 |
PCR Grade Water | 20 | 100 | 180 | 220 | 300 | 320 | 420 |
DMSO | 1.5 | 7.5 | 13.5 | 16.5 | 22.5 | 24 | 31.5 |
Betaine (5 M) | 13 | 65 | 117 | 143 | 195 | 208 | 273 |
Sense Primer (10 µM) | 1.5 | 7.5 | 13.5 | 16.5 | 22.5 | 24 | 31.5 |
Antisense Primer (10 µM) | 1.5 | 7.5 | 13.5 | 16.5 | 22.5 | 24 | 31.5 |
Template DNA | 1 | 5 | 9 | 11 | 15 | 16 | 21 |
Phusion DNA Polymerase (1 U/µL) | 0.5 | 2.5 | 4.5 | 5.5 | 7.5 | 8 | 10.5 |
Total Volume | 50 | 250 | 450 | 550 | 750 | 800 | 1050 |
*amounts for each are in uL
Q5 2x Master Mix
Q5 Master Mix recipe available on New England Biolabs Website
DreamTaq 2x Master Mix
PCR Component | Number of PCR Reactions* | ||||||
1 | 5 | 9 | 16 | 21 | 26 | 31 | |
2X Master Mix (DreamTaq) | 5 | 25 | 45 | 80 | 105 | 130 | 155 |
Forward Primer (10 µM) | 0.5 | 2.5 | 4.5 | 8 | 10.5 | 13 | 15.5 |
Reverse Primer (10 µM) | 0.5 | 2.5 | 4.5 | 8 | 10.5 | 13 | 15.5 |
Betaine (5 M) | 2.6 | 13 | 23.4 | 41.6 | 54.6 | 67.6 | 80.6 |
DMSO | 0.5 | 2.5 | 4.5 | 8 | 10.5 | 13 | 15.5 |
Water | 0.9 | 4.5 | 8.1 | 14.4 | 18.9 | 23.4 | 27.9 |
Total Volume | 10 | 50 | 90 | 160 | 210 | 260 | 310 |
*amounts for each are in uL
Step | Temp | Time |
Initial Denaturation | 98°C | 30 seconds |
25 cycles | 98°C 45-72°C 72°C | 5-10 seconds 10- 30 seconds 60- 120 seconds per kb |
Final Extension | 72°C | 5-10 minutes |
Hold | 4°C |
DNA Gel Electrophoresis
Preparing Gel
- Weigh 1 g of ultrapure agarose. Add to 100 mL of 1x TAE buffer solution
- Microwave and swirl periodically until fully dissolved
- When almost cooled, add 2 µL of ethidium bromide
- Place combs, desired side down, and pour solution
Running Gel
- Pour 1x TAE buffer till entire gel is covered
- Load samples
- In the first well, load 10 µL of desired DNA ladder
- Amount of PCR product depends on comb used to create well
- 50 µL of pcr product
- 10 µL of 6x loading dye
- Mix by pipetting
- Plug in the electrodes in a Negative to Positive direction
- Check for bubbling at the negative end (top)
- Run for 25 minutes at 125V
- Turn off electrodes before removing cover
- Look at gel under UV transilluminator
- Compare size of band against ladder
- Use a razor blade to cut and transfer band of DNA to a clean, pre-labeled eppendorf tube
- Repeat for each well.
- Freeze at -20°C or continue onto gel purification protocol
- Dispose of gel into specified ethidium bromide waste
Gibson Cloning
- Prepare Gibson Assembly Master Mix as follows:
- Store at -20°C in 15 µL aliquots
Component | Volume (uL) |
5X ISO Buffer | 320 |
10 U/µL T5 exonuclease | 0.64 |
10 U/µL Phusion polymerase | 20 |
40 U/µL Taq ligase | 160 |
Water | 699 |
Total | 1200 |
- Thaw a 15 µL aliquot of the Gibson assembly master mix and keep on ice until use
- Measure the DNA concentration (ng/µL) of each assembly piece
- Add 100 ng of the linearized vector backbone and equimolar amounts of the other assembly pieces to the thawed 15 µL master mix in a 20 µL total volume assembly reaction mixture as follows:
Linearized vector backbone (100 ng) | ||
+ | Each additional assembly piece (to equimolar with backbone) | |
+ | 15 µL | Gibson assembly master mix |
+ | dH2O to | |
20 µL |
- Incubate the assembly reaction at 50°C for 60 minutes
- Store at -20°C
- Use for transformations into DH5α competent cells
Transformation
Transformation
- Combine
- 20 µL 5x KCM
- + x µL DNA (~10 ng or 5uL of Gibson reaction product)
- + ddH2O to 100 µL
- Add 100 µL competent cells, mix gently and incubate on ice for 20 minutes
- DH5α for Gibson products and plasmid storage
- Bl21 for Protein Expression
- Rosetta for rare codon protein expression
- Heat shock at 42°C for 90 sec
- Place on ice for 1 minute
- Add 400-600 µL LB media to cells
- Shake for 45 min to 1 hour at 37°C
- Plate 50-250 µL cells
Miniprep
- Transfer 1.5 mL of culture eppendorf tubes
- Centrifuge for 1 min to pellet cells
- Discard supernatant
- Repeat 2 times
- Using the ThermoFisher Genejet kit
- Add 250uL of resuspension buffer to the pellet and resuspend
- Pipet up and down to fully resuspend cells
- Should become cloudy
- Add 350uL of Lysis solution
- Invert 4-6 times
- Should turn clear and viscous
- Add 350uL of neutralization solution
- Invert 4-6 times
- White precipitate should form
- Centrifuge for 5 minutes at 14.7 xg to pellet cell walls and chromosomal DNA
- Transfer supernatant to centrifugal filter tubes
- Centrifuge for 1 minute
- Discard flow through
- Continue until all of the sample has been filtered
- Elute with sterile water into clean, labeled eppendorf tube.
- 50mL
Plasmid Screening (Colony PCR)
PCR Component | 1 | 5 | 9 | 16 | 21 | 26 | 31 |
2X Master Mix (DreamTaq) | 5 | 25 | 45 | 80 | 105 | 130 | 155 |
Forward Primer (10 µM) | 0.5 | 2.5 | 4.5 | 8 | 10.5 | 13 | 15.5 |
Reverse Primer (10 µM) | 0.5 | 2.5 | 4.5 | 8 | 10.5 | 13 | 15.5 |
Betaine (5 M) | 2.6 | 13 | 23.4 | 41.6 | 54.6 | 67.6 | 80.6 |
DMSO | 0.5 | 2.5 | 4.5 | 8 | 10.5 | 13 | 15.5 |
Water | 0.9 | 4.5 | 8.1 | 14.4 | 18.9 | 23.4 | 27.9 |
Total Volume | 10 | 50 | 90 | 160 | 210 | 260 | 310 |
Step | Temp | Time |
Initial Denaturation | 98°C | 30 seconds |
35 cycles | 98°C 45-72°C 72°C | 5-10 seconds 10- 30 seconds 60- 120 seconds per kb |
Final Extension | 72°C | 5-10 minutes |
Hold | 4°C |
Microscope preparation (basic compound microscope)
- Add 10uL of sterile water to a pcr tube
- Use a pipet-tip to transfer some cells from plate to the water by lightly scraping cells off the plate and swirling it in the water
- Plate 2 µL of cells onto a microscope slide, place a slide cover over sample
Protein Expression
Expression Optimization Protocol
Day 1:
- Inoculate specified volume* of LB media + antibiotic overnight at 37°C in a 150 mL flask with BL21 cells with desired plasmid
* 10 mL of overnight culture per 1 L of expression culture
Day 2:
- Inoculate large culture of LB media + antibiotic with overnight culture
- Ensure culture flask is at least 3x the volume of the culture to ensure adequate aeration
- Grow to OD600 of 0.4 to 0.6
- Take a 1 mL sample of the culture, centrifuge at 21,000 xg for 1 minute, and resuspend in 50 µL of denaturing sample buffer. Set aside for expression verification gel.
- Induce expression with 1mM [IPTG]final
- [IPTG]final = 1 mL of 1M IPTG per liter of large culture
- Shake at 37°C for 4 hours
- Take a 1 mL sample of the culture, centrifuge at 21,000 xg for 1 minute, and resuspend in 50 µL of denaturing sample buffer. This will be run with the 1 mL sample from earlier.
- Heat both 1mL samples at 75-100 °C for 5-10 minutes.
- Vortex and centrifuge again at 21,000 xg for 5 minute before loading on a gel to test expression
- Spin large cultures down at 4000 xg for 15 minutes
- Pour off liquid into sink
- Repeat for all of the same culture into the same bottle
- Resuspend in resuspension buffer.
- Pellet resuspended cells into 50 mL tubes (conicals) at 4000 xg for 10 minutes
- Pour off liquid and freeze at -80°C until ready for purification
Protein Purification
Purification Optimization Protocol
Day I
- Resuspend cells in the 20-100 mL Lysis Buffer and scrape, vortex, or pipette up and down.
- Lyse the cells using a Sonic-Dismembrenator or Emulsiflex and collect the resulting solution.
- Clarify the lysate by centrifugation at 20,000 xg at 4 °C for 90 minutes.
- Take a small sample of the pellet and store in denaturing SDS buffer
- Take a small sample of the supernatant and store in denaturing SDS buffer
- Incubate supernatant with Ni-NTA resin for 2 hours
- Transfer supernatant + Ni-NTA resin into a size-exclusion column and allow to drain
- Take a small sample of the flowthrough and store in denaturing SDS buffer
- Wash the column with Wash Buffer 3 times.
- Take a small sample of the wash sample and store in denaturing SDS buffer
- Elute the protein using Elution Buffer 1 times.
- Take a small sample of the wash sample and store in denaturing SDS buffer
- Analyze protein purity by SDS-PAGE.
- Run all of the samples taken prior to check for presence of protein.
Gel Staining
Coomassie Staining
- Microwave the gel in deionized water for 30 seconds. Repeat three times to remove any residual SDS.
- Soak the gel in enough Coomassie Safe Stain to cover the surface, microwave for 30 seconds, and let sit for 10 minutes, shaking occasionally.
Coomassie Destaining
- Pouring out theCoomassie stain
- Rinse the gel with deionized water
- Cover the gel in deionized water and microwave the gel for 30 seconds
- Leave on orbital shaker until gel is destained
- Add a kimtech wipe to absorb residual coomassie stain.
Diafiltration -Buffer Exchange
- Load elution into diafiltration column with the column facing downwards
- Centrifuge at 4000 xg for 45 min at 4°C to concentrate sample
- Add 2 mL splicing buffer to the column.
- Centrifuge at 4000 xg for 45 min at 4°C to concentrate and exchange buffers
- Remove filter collection tube and invert column
- Centrifuge at 1000 xg for 2 min at 4°C to elute sample
- Store at -20°C
Intein Assays
Purified and Dialyzed Protein Assays
- Thaw the splicing buffer dialyzed proteins on ice
- Combine equimolar concentrations of each protein
- Shake at 37°C
- Take 2 uL aliquots at designated times and store samples in denaturing SDS buffer
- Run gel
- Run with individual proteins as controls
- Stain gel with Coomassie Stain
Lysate Splicing Assays
- Lyse expressed pellets through physical means (sonication, emulsiflex) into splicing buffer
- Set aside 2uL of lysate in denaturing SDS to be run on the gel
- Optional centrifugation to clarify lysates (depends on protein solubility)
- Combine each protein lysate
- Shake at 37°C
- Take 2 uL aliquots at designated times
- Run gel
- Run with individual lysates as controls
- Stain gel with Coomassie Stain or HIS- Stain