Team:IISc-Bangalore/Protocols

  1. Cell Protocols
  2. Molecular Biology
  3. Assays
  4. Reagents

Cell-Based Protocols

Preparation of competent cells — The TSS method

For E. coli cells to take up a foreign plasmid, they have to first be made competent - their cell walls must be weakened and made permeable to incoming DNA. This is traditionally done using divalent cations, which mask the negative charge of the phospholipid membrane, allowing the negatively-charged DNA to approach the cell without significant repulsion. The Transformation and Storage Solution (TSS) contains Mg++.

Day 1
Step Description Rationale
1 Prepare a primary inoculum of E. coli in 5 mL LB in a test tube. -
2 Incubate overnight at 37°C, 170 rpm. -
Day 2
3 Inoculate 1 mL overnight culture in 100 mL LB medium (1% inoculum) This protocol yields one aliquot of competent cells per mL of culture. Depending on how many aliquots of competent cells you wish to make, you can vary the volume of LB medium used.
4 Incubate at 37°C, 170 rpm until the OD reaches 0.4 (~1 h 45 min for E. coli DH5α cells) This is the early exponential phase; cells are physiologically ideal for the preparation of competent cells.
From this point, cells should always be placed in the cold (below 4°C), all buffers should be ice-cold, and all plasticware/glassware should be pre-chilled.
5 Place the culture at 4°C for 45 min -
6 Spin down the culture at 10000 rpm, 10 min, 4°C -
7 Resuspend the cell pellet in 1 mL ice-cold TSS buffer -
8 Spin down the culture at 10000 rpm, 10 min, 4°C -
9 Make 50-100 µL aliquots in chilled microfuge tubes, snap-freeze in liquid nitrogen and store at -80°C for long-term storage -

Transformation of Chemically Competent E. coli: The Heat-Shock Method

Step Description Rationale
1 Thaw the competent cells on ice for 20 min -
2 Add 1-5 µL of plasmid DNA (10 pg - 100 ng) directly into the competent cells. -
3 Gently flick the microfuge tube to mix the cells and the DNA. -
4 Incubate on ice for 30 min. -
5 Heat-shock the cells at 42°C for ~45 s The optimal heat-shock duration varies from 30 s to 90 s depending on strain, batch, and method of preparation. In our experiments, 45 s gave us a high enough transformation efficiency to proceed.
6 Place the cells on ice for 5 min -
7 Add 950 µL SOC medium. -
8 Incubate at 37°C, 220 rpm for 1-2 h. The optimal incubation time varies depending on strain and antibiotic resistance marker used for selection. This step allows the few transformants to replicate and increase their plasmid number in the following generations.
9 Spread plate 100 µL of the culture on a selection plate. -
10 Spin down the remaining culture at 5000 rpm, 10 min, resuspend in ~100 µL medium, and spread plate on a selection plate. -
11 Incubate plates overnight at 37°C until transformant colonies are seen. -

Molecular Biology

Isolation of Plasmid DNA from Transformants: The Miniprep Protocol

Day 1
Step Description
1 Inoculate one colony of the desired transformant in 5 mL of LB medium in a test tube.
2 Incubate overnight at 37°C, 170 rpm.
1 Spin down 1.5 mL of the overnight culture in a 1.5 mL microfuge tube at 5000 rpm, 10 min. Discard the supernatant. Repeat until all 5 mL has been pelleted.
2 Resuspend the cell pellet in 200 µL alkaline lysis solution I using a micropipette.
3 Keep the microfuge tube on ice for 5 min.
4 Add 400 µL alkaline lysis solution II along the sides of the microfuge tube.
5 Very gently invert the microfuge tube three times.
6 Keep the microfuge tube on ice for 30 s.
7 Add 300 µL alkaline lysis solution III.
8 Gently invert the microfuge tube three times.
9 Keep the microfuge tube on ice for 1 min.
10 Spin at 13000 rpm for 10 min.
11 Transfer the supernatant to a 2 mL microfuge tube.
12 Add RNase A (5 µg/mL) to the supernatant, and incubate at 65°C for 30 min.
13 Add an equal volume of chloroform to the microfuge tube.
14 Spin at 13500 rpm, 10 min, 4°C
15 Carefully transfer the upper aqueous layer to a new microfuge tube, avoiding white particles of protein at the water-chloroform interface.
16 Repeat Steps 13, 14, and 15.
17 Add an equal volume of ice-cold isopropanol.
18 Incubate on ice for 30 min.
19 Centrifuge at 14500 rpm, 30 min, 4°C.
20 Discard supernatant and add 500 µL ice-cold 70% ethanol.
21 Centrifuge at 14500 rpm, 30 min, 4°C.
22 Discard supernatant, mark the position of the pellet, and leave the excess ethanol to dry.
23 Resuspend the DNA pellet in 20 µL MilliQ.

PCR

Transfer all the mention components to the PCR eppendorfs. Gently spin them down to ensure that all the components have properly mixed.

Component 25 µl Reaction 50 µl Reaction Final concentration
10x Standard Taq Reaction buffer 2.5 µl 5 µl 1x
10mM dNTPs 0.5 µl 1 µl 200uM
10um Forward Primer 0.5µl 1µl 0.2µM (0.05-1µM)
10 µM reverse Primer 0.5µl 1µl 0.2µm (0.05-1µM)
Template DNA variable variable <1000ng
Taq DNA Polymerase 0.125µl 0.25µl 1.25 units/50µl PCR Reaction
Nuclease-free water to 25µl to 50µl

Transfer PCR tubes from ice to a PCR machine with the block preheated to 95°C and begin thermocycling

Thermocycling conditions for routine PCR

Step Temp Time
Intial denaturation 95 degrees 30 seconds
30 cycles 95 degrees
45-68 degrees
68 degrees 		15-30 seconds
15-60 seconds
1 minute/kB
Final extension 68 degrees 5 minutes
Hold 4-10 degrees

Colony PCR

Transfer all the mention components to the PCR eppendorfs. Gently spin them down to ensure that all the components have properly mixed.

Component 25 µl Reaction 50 µl Reaction Final concentration
10x Standard Taq Reaction buffer 2.5 µl 5 µl 1x
10mM dNTPs 0.5 µl 1 µl 200µM
10um Forward Primer 0.5µl 1µl 0.2µM (0.05-1µM)
10 µM reverse Primer 0.5µl 1µl 0.2µM (0.05-1µM)
Taq DNA Polymerase 0.2µl 0.4µl
Water + colony 19.3µl 38.6µl

Transfer PCR tubes from ice to a PCR machine with the block preheated to 95°C and begin thermocycling

Thermocycling conditions for colony PCR

Step Temp Time
Intial denaturation 95 degrees 30 seconds
30 cycles 95 degrees
45-68 degrees
68 degrees 		15-30 seconds
15-60 seconds
1 minute/kB
Final extension 68 degrees 5 minutes
Hold 4-10 degrees

DNA purification

Chloroform extraction

Main aim of this procedure is to separate Proteins from the DNA samples. Usually this is required if you further plan to perform restriction digestions or ligations with the DNA as the activity of the enzyme is affected by the presence of the contaminants.

Step Procedure
1. Take the DNA sample and add equal amount of chloroform to it.
2. Spin down everything at 10000 rpm, 10 min, 4ºC
3, Take the top aqueous layer. To it add equal amount of chloroform. (usually two chloroform washes are done, but if you have very less DNA concentration and you are OK with some contamination, only one wash is good enough, try not to take the chloroform layer when you are separating the aqueous layer.
4. Spin down everything at 10000prm, 5 min, 4ºC
5. Take the top aqueous layer. To it add equal amount of isopropanol. Keep this mixture on ice for 30 min. ( it is recommended to do this step in a 1.5mL Eppendorf so that the pellet you will be looking for on centrifugation will be easily visible.
6. Spin down the mixture at 14500rpm, 30 min, 4ºC
7. You should observe a pellet after the end of centrifugation.
8. Discard the supernatant and dry the pellet at 65ºC in a hybridization oven or at 37ºC in the incubator.
9. Re-suspend the pellet in 20 uL of miliQ water. The pellet might become invisible when drying, no need to panic.
10. Take nano-drop readings after this.

Gel purification

Step Procedure Rationale
1. Excise the DNA band of interest using an ethanol cleaned razor blade or a scalpel on a transilluminator Minimize gel volume by cutting as small a slice as possible. Use of long wave transilluminator and short handling time will lead to better quality DNA
2. Weigh the gel slice in a microcentrifuge tube. Add 3 volumes (µl) of buffer GB to 1 volume (mg) of gel
3. Indubate at 50 degree Celcius until the agarose gel is completely melted (5-10 min) To help effective dissolving of the gel, vortex every 2-3 minutes during incubation
4. AFter the slice has dissolved completely, check that the colour of the mixture is yellow (similar to buffer GB) If the colour of the mixture becomes brown or purple, add 10ul of 3M Sodium Acetate, pH 5.0 and mix. The colour of the mixture will turn to yellow
5. (Optional) Add 1 gel volume of the isopropanol to the sample and vortex to mix Do not centrifuge at this step
6. Transfer the mixture to a SV column. Centrifuge for 1 min at 10,000x g and above (>12,000 RPM). Discard the pass through and res-insert the SV column intro the collection tube IF the mixture volume is greater than 700µl, apply the mixture twice; apply 700ul of the mixture, dpin down, discard the pass through,re-insert empty collection tube, and repeat until all the mixture has been applied to the SV column
7. (Optional) Apply 500µl of buffer GB to the SV column. Centrifuge for 30 seconds. Discard the passthrough and re-insert the SV column into the collection tube. This is to further remove any traces of agarose and required only for direct use of DNA in very sensitive applications
8. Add 700µl of buffer NW to the SV column. Centrifuge for 30 seconds. Discard the pass-through. Reinsert the SV column into the collection tube If the purified DNA will be used for salt sensitive applications, let the SV column stand for 5 min after addition of buffer NW, making some amount of wash buffer flow through the column by gravity before centrifugation
9. Centrifuge for an additional 1 min to remove residual wash buffer. Transfer the SV column to a new 1.5 ml centrifuge tube. If residual ethanol still remains, centrifuge again for 1 min at full speedbefore transferring to a new 1.5 ml microcentrifuge tube
10. Apply 50µl of buffer EB or ddH2O to the center of the membrane in the SV column, let stand for 1 min and centrifuge for 1 min Ensure that buffer EB or distilled water is dispensed directly onto the centre of the SV column for optimal elution of DNA. For larger fragment (>5 kB), used pre-warmed elution buffer (70 degree celcius) for best efficiency. For long term storage, eluting in buffer EB (10 mM Tris Cl, pH 8.5 or TE, pH 8.0 and storing under -20 degree celcius is recommended.

Restriction digestion

Step Procedure Rationale
1. Select the Restriction enzymes you need to digest your plasmid with.
2. Select the appropriate buffer for the working of the enzyme. If you are doing double digest, then you need to find a buffer which gives optimal activity with both the enzymes. If you are not able to find any such buffer, then you have to proceed with sequential digestion.
3. A typical restriction digestion reaction could look like this:
  • 1 µg DNA
  • 1 µl each of Restriction enzymes
  • 5 µl 10x buffer
  • 3 µl 10x BSA(if recommended, usually most of the enzymes nowadayshave BSA in them)
  • x µl dH2O (bring it up to 50µl)
4. MIx well by gently pipetting
5. Incubate at appropriate temperature (usually 37 °C) for 1 hour. You can also do an overnight digestion by incubating overnight, just add less amount of enzyme.
6. After the digestion, run a 1% agarose gel to check if the digestion has worked.
  • One can also proceed to gel elution to get the specific band required.
  • To purify the plasmid for further usage perform Chloroform extraction, since the efficiency of Gel elution is very low.
  • For sequential digestion, if need to be done, heat inactivate the enzyme by incubating at 70°C for 10-15min
  • If your enzyme did not cut, check to make sure that it isn’t methylation sensitive. Plasmids grown in Dam or Dcm methylase positive strains will be resistant to cleavage at certain restriction sites.

Ligation

The volume of vector DNA and insert DNA used in the ligation will vary depending on the size of each and their concentration. However, for most standard cloning (where the insert is smaller than the vector) a 3 (insert):1 (vector) ratio will work just fine

Step Procedure
1. Combine the following in a PCR or an Eppendorf tube:
  • 25ng Vector DNA
  • 75ng Insert DNA
  • Ligase Buffer (1μL/10μL reaction for 10X buffer, and 2μL/10μL reaction for 5X buffer)
  • 0.5-1μL T4 DNA Ligase
  • H20 to a total of 10μL
2. Incubate at room temperature for 2 hrs or overnight at 16°C>
3. Proceed with bacterial transformation. You can use 10 µL of ligation mixture with 100 uL of competent cells. There is no need to purify the ligation mixture.

Running the controls

Control Ligase Interpretation
Uncut Vector - Checks viability of competent cells and verifies the antibiotic resistance fo the plasmid.
Cut Vector - Background due to uncut vector
Cut Vector + Background due to vector recircularization - most useful for phosphatase treated vector

SDS Page

Before you go for the electrophoresis part, make sure the target protein is dissolved in the liquid phase, for this you sonicate or lyse the cells, once the protein is released, centrifuge to separate the supernant and the pellet. Make sure there is no guanine hydrochloride so that SDS is not precipitated.

Step Procedure
Resolving Gel
1. Set the casting frames. Clamp them properly, make sure the spacers and the comb is of the same size so that you get good wells.
2. Make 1% agarose gel to seal the bottom of the cast.
3. Make the Resolving gel, once you add TEMED, the polymerization starts. So quickly add the gel into the cast. Add appropriate amount. Keep some aside to keep a check on, if the gel has solidified.
4. Add some water to the gel so that you get a good horizontal gel.
Stacking Gel
1. Once the resolving gel has solidified, you can remove the water layer
2. Make the stacking gel solution. Place the comb properly.
3. Once you add TEMED, add the gel to the cast. Add little more than required as the volume of the gel decreases dues to polymerization.
4. Check solidification of the gel. Once it has solidified, keep it in the tray and add the electrophoresis buffer to it.
Sample preparation
1. Add 1:6 ratio of 6X Loading dye to the protein sample. Incubate it at 95ºC for 10min.
2. Load samples properly to the wells. Make sure you also run a ladder, to look for the protein of interest.
Staining and destaining
1. Once you think the gel has ran enough such that the ladder has properly resolved.
2. Put the gel carefully in the staining solution. You can remove the stacking gel part and also make a cut to the opposite end of the ladder for easy identification.
3. You can keep the gel in the staining solution on a gel rocker (if available) over night.
4. After that, make a fresh destaining solution (recommended). You should replace the solution at appropriate intervals for better destaining.
5. Then view the gel under white light.

Our Assays

Scanning Electron Microscopy (SEM) assay

Scanning electron microscopy was done by taking samples of treated gas vesicles at various concentrations and the images were analyzed. The protocol is as follows,

Step Description Remarks
1 Using double sided carbon tape, stick multiple circular glass slides on a metal disc. -
2 Load 10µl of the samples on each glass slide. -
3 Put the disc under a vacuum desiccator until all the liquid dries up leaving only the dried gas vesicles on the slide. If the gas vesicles are suspended in PBS, large salt crystals and salt flakes might be visible under the microscope (see image). If a large amount of gas vesicles are available, dialysis can be performed to get rid of the salt crystals.
4 Apply a gold sputter coating of 10nm thickness to the samples. -
5 Load the disc in the Scanning electron microscope to be imaged at various magnifications. -

Appearance of salt in dried SEM samples

Dynamic Light Scattering (DLS) assay

Dynamic Light Scattering (DLS) sometimes also called Photon correlation spectroscopy is a technique that allows the estimation of the size of nanoparticles in a suspension by monitoring the scattering as a particle undergoes brownian motion. It proved to be extremely useful in our experiments due to the fact that it directly outputs the effective hydrodynamic size (the diameter of a hard sphere which diffuses in a similar manner as the particle of interest) which is a determining factor in our model. We expect the sizes of the particles to increase after flocculation and the results can be seen in the results section of this wiki.

The protocol is as simple as taking the same sample at various dilutions and putting it in a cuvette to be analyzed by the DLS machine (Zetasizer nano). The exact amounts that were required in an experiment are given next to the data that was obtained.

Spectrophotometry assay

A method to check the effective flotation was devised by using a spectrophotometer to record the OD of a dilute gas vesicle suspension at 500nm. As the gas vesicles float to the surface over time, the OD reduces giving a decreasing curve. The thermal fluctuations (Brownian motion) at room temperature are still significant and hence the curve is not as smooth as we would expect for a heavy particle. However, the curves do provide some interesting insights into how advective and diffusive transports compete for small particle sizes. The exact protocol for flotation spectrophotometry is straightforward.

Step Description Remarks
1 Turn on the spectrophotometer for time analysis and set it to take readings for 2 hours at 0.1min intervals. -
2 Add 2mL PBS to a cuvette for use as a blank. -
3 Dilute the gas vesicle stock to 2mL to be used in the cuvette. In all our experiments the effective gas vesicle concentration in the final suspension was ~15ng/ul.
4 Invert the cuvettes slowly a number of times and put them in their respective slots in the spectrophotometer PBS for blank, Sample in active slot
5 Start the two hour timed run -

Reagents

TSS buffer

Reagent Volume Final concentration
2x LB medium 5 mL 50% (v/v)
25% PEG 3350 (w/v) 4 mL 10% (w/v)
DMSO 0.5 mL 5% (v/v)
2M MgCl2 0.5 mL 0.1 MgCl2
Filter sterilize the TSS buffer using a 0.22 µm filter.

Alkaline lysis solution I

Reagent Volume Final concentration
glucose - 50 mM
Tris-Cl (pH 8.0) - 25 mM
EDTA (pH 8.0) - 10 mM
MilliQ water - -
Autoclave the solution at 121°C, 15 psi, 15 min to sterilize

Alkaline lysis solution II

Prepare a fresh solution just before use.
Reagent Volume Final concentration
1 N NaOH 2 mL 0.2 N
10% SDS (w/v) 1 mL 1% (w/v)
MilliQ water 7 mL Up to 10 mL

Alkaline lysis solution III

Reagent Volume Final concentration
5 M KOAc 60 mL 3 M
glacial acetic acid 11.5 mL 11.5% (v/v)
MilliQ water 28.5 mL Up to 100 mL
Autoclave the solution at 121°C, 15 psi, 15 min to sterilize

Stacking gel - 5ml 5%

Reagent Volume
H2O 3.4 ml
30% acrylamide 830 µl
1M Tris (pH 6.8) 630µl
10% SDS 50 µl
10% APS 50 µl
TEMED 5 µl

5x loading buffer

Reagent Amount
SDS 10% w/v
ß-mercaptoethanol or dithiothreitol 10mM
Glycerol 20% w/v
Tris (pH 6.8) 0.2M
Bromophenol Blue 0.05% w/v