Team:GeorgiaTech/Protocols
Protocols
Preparing Cell Solid and Liquid Media
Materials
|
Tools
|
Equipment
|
Protocol
- LB Miller Media
- 10 g NaCl / 1 L DI water
- 10 g peptone / 1 L DI water
- 5 g yeast extract / 1 L DI Water
- LB Miller Agar
- 9.25 g agar mix / 250 mL water (use a 1 L Erlenmeyer flask)
- Sterilization
- Place a stir bar in the Erlenmeyer flask containing the agar solution
- Place all glassware in a secondary container
- Loosely cover all glassware with double layer foil and apply autoclave tape
- Autoclave on LIQ 20 cycle (1 hour)
- Cool the contents to 50-60 C (30 minutes)
- Distribute Agar to Petri Dishes
- Add 250 uL of antibiotic (100 mg/mL) into agar stock
- Over a bunsen burner flame, place 10 mL of agar stock in each petri dish using serological pipet and pump
- Wait for the agar to solidify at room temperature (30-45 minutes)
Chemical Plasmid Transformation, Plating, and Inoculation
Materials
|
Tools
|
Equipment
|
Protocol
- Place 25 uL of chemically competent E. coli cells in each microcentrifuge tube
- Dilute plasmid to 50 ng/uL and place 1 uL of diluted plasmid in each microcentrifuge tube
- Cool the tubes on ice (0-4 C) for 15 minutes
- Heat the tubes in a hot water bath at 42 C for 1 minute (heat shock)
- Over a Bunsen burner flame, add 150 uL of LB media to each tube
- Place tubes in an Erlenmeyer flask and incubate in a shaker at 37 C for 45 minutes
- Plate 10 uL of cells from each tube onto an LB agar dish
- Sterilize an agar spreader over a Bunsen burner flame and spread the cells around the gel
- Store the plates upside down at 37 C overnight
After 24 hours:
- Sterilize works surface and gloves with 70% ethanol. Use the Bunsen burner to flame the exterior of the serological pipet barrel
- Draw LB media and dispense ~5 mL in each empty cell culture tube
- Sterilize the loop by dipping in ethanol and passing it over the Bunsen burner flame
- Cool the loop on the edge of an agar plate and scoop one bacterial colony out of the plate using the sterilized loop
- Mix the bacterial colony into the LB contained in one of the cell culture tubes
- Add 5 uL of ampicillin (100 mg/mL) to each cell culture tube
- Incubate the tubes in a shaker at 37 C overnight
Plasmid Purification
Adapted from QIAprep Spin Miniprep Kit DNA Plasmid Isolation handbook
Materials
|
Tools
| Equipment
|
Protocol
- Store P1 at 4 C, P2 at 37 C, and N3, PE, and EB at room temperature
- Store 1 mL of plasmid from cell culture tubes (see plasmid transformation) in a 1.5 mL microcentrifuge tube
- Spin down plasmid (in LB) at 13000 rpm for 2 minutes to obtain a pellet
- Remove and discard supernatant by decanting or pipetting
- Resuspend pellet in 100 uL of P1 buffer. Perform this in ice.
- Add 100 uL of P2 (lysis) buffer and invert 4-6 times to mix. Do not allow lysis to occur for more than 5 minutes.
- Add 140 uL of N3 buffer and invert 4-6 times to mix.
- Spin at 13000 rpm for 10 minutes.
- Remove supernatant and transfer to a spin column
- Spin 13000 rpm for 1 minute
- Discard flow through and add 300 uL of PE (wash) buffer
- Spin at 13000 rpm for 1 minute
- Discard flow through and spin at 13000 rpm for an additional 1 minute
- Discard flow through and transfer the spin column to a clean 1.5 mL centrifuge tube
- Add 50 uL EB buffer and let stand for 1 minute
- Spin at 13000 for 1 minute
- Flow through contains plasmid
Characterization of Purified Plasmid
Materials
|
Tools
| Equipment
|
Protocol
- Concentration determination by NanoDrop spectrophotometer
- Dilute plasmid with EB as necessary to achieve absorbance readings around 0.3
- Our plasmids required 4 to 6-fold dilutions; we mixed 5 uL of plasmid with 15 uL of EB
- Clean the spectrophotometer with 2uL of DI water, dab with a KimWipe
- Blank the spectrophotometer with 2 uL of EB, dab with a KimWipe
- Load 2 uL of diluted plasmid and record the absorbance at 260 nm (corresponding to nucleic acids) and the plasmid concentration (ng/uL)
- Confirmation of DNA size by gel electrophoresis
- Determine gel electrophoresis parameters, including voltage and run time, quantity (mass) of DNA to be loaded, and agarose concentration
- Prepare agarose gel
- Prepare 1% w/v solution of agarose (for small plasmids) in the appropriate amount of TAE buffer
- An 8-lane rig requires approximately 20 mL and a 12-lane rig requires approximately 40 mL gel solution
- Microwave the mixture until the solution is transparent and bubbling
- Add 2 or 4 uL (0.1% v/v) of SYBR safe dye and stir
- Insert the gel inset and comb into the mold and pour the gel solution over the inset
- Chill at 4 C for 10 minutes to speed up gel solidification; otherwise, gel can stand at room temperature until solidified
- Prepare plasmid mixtures to be loaded into the gel
- Determine the required volume of plasmid that contains the desired mass of DNA. Apply plasmid to a sheet of parafilm.
- Add 1.1 uL of dye to the plasmid (⅙ of the mixture’s total volume)
- Subtract the combined volume of plasmid and dye from 6.6 uL and add that amount of water to each mixture. (i.e. “top off” the mixture to 6.6 uL with water)
- Load mixtures into gel
- Remove the gel from 4 C and set the gel in a gel electrophoresis rig filled with TAE buffer
- Transfer 6 uL of each plasmid mixture to one of the wells
- Transfer 2 uL of ladder reference standard DNA solution to one of the wells
- Transfer 6 uL of blue dye blank to each of the remaining empty wells
- Record the contents of each lane in the well
- Run gel by applying desired voltage to the mini gel migration tank. After the desired run time has elapsed, turn off the voltage and remove the gel
- Characterize the gel on the Omega Lum G
PCR with Primer Flanking
Materials
|
Tools
| Equipment
|
Protocol
- In PCR tubes, combine 50 uL 2x Master Mix, 10 uL forward primer, 10 uL reverse primer, 100 ng of plasmids DNA, and enough deionized water to total 100 uL total.
- Place the PCR tubes in the PCR thermocycler and set the extention duration according to the length of the gene sequence (200-400 bp: 30 seconds; 2000 bp: 2 minutes)
- After 36 cycles of PCR, remove the samples from the PCR thermocycler and store the products at 4 C
Gel Purification
Materials
|
Tools
| Equipment
|
Protocol
Gel Electrophoresis Separation
- Prepare 1% agarose gel with 0.1% SYBR Safe dye and pour it over the inset and comb
- Allow the gel to solidify at room temperature
- Remove the comb and add all of the PCR product (100 uL) for each gene to each well. The PCR product contains dye (incorporated in the master mix) so no additional dye is needed
- Add 6 uL of DNA ladder to one of the lanes
- Run the gel at 135 V for 30 minutes
- Image the gel under the Omega Lum G Imaging instrument for documentation purposes
- View the gel under UV light and locate the appropriate band in each lane
- Use a razor blade to cut around the glowing bands and place each in a clean 1.5 uL centrifuge tube
Purification
- Add 600 uL of gel extraction buffer to each tube and place in a 70 C heating block
- Rapidly remove the tubes one at a time and mix each tube by inversion, placing each tube back in the heating block while it is not being inverted, until the gel is completely dissolved (about 3 minutes)
- Add 200 uL of isopropanol to each tube and mix by inversion
- Transfer each gene solution into a spin column
- Centrifuge at 10,000 rpm for 10 seconds and discard the flowthrough
- Add 750 uL PE buffer
- Centrifuge at 10,000 rpm for 10 seconds and discard the flowthrough
- Centrifuge again at 10,000 for 1 minute to ensure that all the PE buffer is removed
- Transfer each spin column to a new 1.5 uL microcentrifuge tube
- Add 50 uL of water (EB can also be used) to each spin column to elute the DNA
- Centrifuge at 13,000 rpm for 1 minute
- The flowthrough contains the purified PCR product
- Analyze in the NanoDrop spectrophotometer
TOPO Ligation
Materials
|
Tools
| Equipment
|
Calculations
- Use a 25 ng (1 uL of 25 ng/uL solution) TOPO vector basis (TOPO vector is 3.9 kb)
- Calculate the mass (and volume) of each insert required for 5-fold molar excess
- For each insert, calculate the dilution factor that brings the “required” volume to 1 uL
Protocol
- Short spin T4 ligase and 5x buffer
- Dilute each insertion gene according to calculations
- In PCR tubes, combine 1 uL of each diluted insertion with 4 uL 5x buffer, 1 uL TOPO vector, 1 uL T4 ligase, and 13 uL DI water
- Flick each tube to mix but do not vortex
- Centrifuge the tubes for 1-2 seconds
- Allow ligation reaction to proceed at 16 C (in the PCR thermocycler) for 6 hours or at 4 C (refrigerator) overnight
Electroporation Plasmid Transformation
Materials
|
Tools
| Equipment
|
Protocol
- Cool the electroporation cuvettes on ice for 5-10 minutes
- In PCR tubes, combine 40 uL of DH5a with 10 uL of plasmid
- Transfer each mixture to an electroporation cuvette
- Use the pulser (1.8 kV for 2 ms) to transform the plasmids in each cuvette
- Plate 10 uL of cells from each cuvette onto LB agar under sterile conditions
- Store the plates upside down at 37 C overnight
Colony PCR
Materials
|
Tools
| Equipment
|
Protocol
- In PCR tubes, combine 10 uL reaction buffer, 1 uL forward primer, 1 uL reverse primer, and 8 uL DI water
- On the agar plate, locate a colony and circle it on the petri dish
- Use a pipet tip to scoop half of the marked colony and mix it into one of the PCR tubes
- Place the PCR tubes in the PCR thermocycler and set the extention duration according to the length of the gene sequence (200-400 bp: 30 seconds; 2000 bp: 2 minutes). Include a boil step in the first cycle for cell lysis
- After 32 cycles of PCR, remove the samples from the PCR thermocycler
- Analyze the products using gel electrophoresis. Bright bands indicate colonies harboring successful TOPO ligations. Dim bands indicate colonies harboring empty TOPO vectors
- Inoculate the other colony halves for the colonies that yielded successful TOPO ligations
TOPO and pET23b Plasmid Digestion
Materials
|
Tools
| Equipment
|
Calculations
- Calculate the volume of 3 ng TOPO ligation product and 2 ng pET23b vector
- Calculate the volume of DI water required to bring the total reaction volume to 100 uL
Protocol
- In PCR tubes, combine 10 uL 10x buffer, 25 uL SfiI, 25 uL NotI, and the calculated volumes of water and TOPO or pET23b plasmids.
- Allow the digestion reaction to proceed at 37 C (in the PCR thermocycler) for 4 hours
- Use gel purification to collect and purify the desired digest fragments
- Excise the smaller fragment (band that traveled further) from the TOPO digestion
- Excise the larger fragment (band that traveled less) from the pET23b digestion
- Measure the concentration of each product using the NanoDrop spectrophotometer
pET23b Ligation
Materials
|
Tools
| Equipment
|
Calculations
- Calculate the volume of pET23b vector required for a 50 ng basis (pET23b vector is 3.6 kb)
- Calculate the mass (and volume) of each insert required for 5-fold molar excess
- Calculate the volume of DI water required to bring the total reaction volume to 20 uL
Protocol
- In PCR tubes, combine 4 uL 5x buffer with the calculated amounts of pET23b vector, insertion gene, and water
- Allow the ligation reaction to proceed at 4 C overnight
- Transform the ligation products into E. coli cells using chemical transformation procedures
Agilent Protein 80 Expression Testing
Materials
|
Tools
| Equipment
|
Protocol
- Transform plasmid into BL21 chemically competent cells and plate the cells for overnight growth
- Inoculate one colony in LB liquid media for overnight growth
- Inoculate 50 uL of the liquid culture in 5 mL of fresh LB with 5 uL ampicillin for 4 hr growth
- Prepare two tubes of each culture; save one of each for the negative control group
- Induce protein expression with 5 uL IPTG into the positive controls and allow 3 hr for full expression
- Load the Agilent Protein 80 chip according to the published protocol that comes with the kit
- Load the Agilent Protein 80 chip into the instrument and run the 2100 expert program
- To interpret the gel electrophoresis results, look for bands that appear in the positive control but not in the negative control of each protein