Team:IISER-Tirupati India/Protocols

Protocols


HPLC/MS

Instrument parameters:
The ‘SHIMADZU’ HPLC system was used for carrying out the experiments. The recorded output data were analyzed using the ‘Lab solution’ software. The C-18 column which is a reverse-phase column was used. The column temperature and flow rate were maintained at 25℃ and 1ml/min respectively. The injection volume was 20µL.

Excreta sample collection and storage:
The excreta samples were collected from a nearby poultry farm having around 5000 chickens using falcon tubes. Methanol was added at the site of the collection itself to ensure biosafety. These samples were then stored at -20℃ until further experimentation.

Standardization of HPLC method for sulfonamides:

  • Sample preparation - To obtain a standard curve for sulfonamides, sulfa drugs were bought from a pharmacy. These sulfa drugs contained sulfadiazine compounds as an active ingredient. The drugs were suspended in methanol, filtered via a PVDF filter to remove the unwanted binders, and then run through the C-18 column.
  • Procedure - Multiple methods were screened from literature and a final protocol was laid out after suitable modifications were incorporated according to the sample. The method set for running the mobile phase involved using a high-pressure gradient system. The solvents used were 0.1% acetic acid (A), pure methanol (B), and pure acetonitrile (C) with varying (v:v) ratios at different time points as mentioned in Table 1.
  • Run time - The total run time was 35 min. The retention times obtained were taken as the standard for future experiments. The eluted fractions were collected and confirmed by MS.

                                   
Table 1
Time(min) Solvent A Solvent B Solvent C
0-15 90 5 5
15-25 82 10 8
25-35 60 28 12


Standardization of HPLC method for sulfonamides:
Ampicillin, purchased from Sigma-Aldrich, was used for standardization of the procedure. The method involved using 0.1% acetic acid and pure acetonitrile as the mobile phase in the 80:20 (v/v) ratio. The mobile phase was run in a high-pressure gradient fashion. The retention time obtained via this method was then taken as the standard for future experiments.
To further confirm the presence of ampicillin, the eluted fraction at the respective retention time was collected, and mass spectrometric analysis was carried out.

HPLC/MS of poultry excreta:
The stored excreta samples were filtered using a PVDF filter and run through the HPLC column following the above mentioned standardized method. Eluted fractions were collected at the respective retention times and the presence of sulfadiazine (SDZ) was confirmed by MS.

Cloning

LB Agar plate preparation with Antibiotic:

  1. Use 15g of LB Agar for 1L of LB medium
  2. Microwave until homogenous and shake carefully every 30s
  3. Let it cool and add Ampicillin to a final concentration of 50µg/ml
  4. Pour 20 mL of liquid in each petri dish, cool until it solidifies


Plasmid Miniprep:
The Monarch plasmid miniprep kit was used for isolating all the plasmids. For the detailed protocol please click here.

PCR DNA Cleanup:
The Monarch PCR DNA cleanup kit was used for isolating all the plasmids. For the detailed protocol please click here.

PCR Mixture:
  1. Q5 High-Fidelity 2X Master Mix: 1X (final concentration)
  2. 10 µM Forward Primer: 0.5µM (final concentration)
  3. 10 µM Reverse Primer: 0.5µM (final concentration)
  4. Template DNA: <1,000ng (for Biobricks and vectors) OR one isolated colony resuspended in 3µL of water (for colony PCR)
  5. Nuclease-Free Water: Till required volume


PCR Conditions:
  1. Initial denaturation at 98°C for 2 minutes
  2. Denaturation at 98°C for 15 seconds, Annealing at 50–72°C for 30 seconds, Extension at 72°C for 30s/kb [This step is repeated 35 times]
  3. Final Extension at 72°C for 5 minutes
  4. Hold at 4°C until removed


Restriction Digestion Mixture (10 µl):
  1. 10X NEB Buffer: 1X (final concentration)
  2. DNA: 0.5µg (final concentration)
  3. Restriction Enzyme: 5 units (0.5µl)
  4. Nuclease-Free Water: Till required volume


Restriction Digestion Conditions:
  1. Pre-heat the Bain Marie to the required temperature
  2. Incubate the mixture at 16°C overnight or at 37°C for one hour
  3. Heat inactivate the restriction enzymes at 80°C for 20 minutes
  4. The digested products are purified using the PCR DNA Cleanup kit


Ligation Mixture (20 µl):
  1. T4 DNA Ligase 10X Buffer: 1X (final concentration)
  2. Vector DNA: 0.020pmol
  3. Insert DNA: 0.060pmol
  4. T4 DNA Ligase Enzyme: 1µl
  5. Nuclease-Free Water: Till required volume


Ligation Conditions:
  1. Pre-heat the Bain Marie to the required temperature
  2. Incubate the mixture at 16°C overnight
  3. Heat inactivate the T4 ligase at 65°C for 10 minutes
  4. This is now used directly for Transformation into competent cells


Transformation:
  1. Turn on the Bain Marie at 42°C
  2. Thaw 50µL of competent cell on ice
  3. Add 3µL of miniprep plasmid to the 50µL bacteria
  4. Chill on ice for 30 minutes
  5. Put it at 42°C for 45 seconds
  6. Put it back on ice for 2 minutes
  7. Add 950µL of SOC or LB medium
  8. Shake incubate at 37°C for 1 hour
  9. Centrifugation at 3000 rpm for 3 minutes
  10. Remove 650µL of supernatant to get a final volume of 350µL
  11. Resuspend the pellet by rubbing the Eppendorf on unregular surfaces
  12. Plate the 350µL on an LB Agar plate containing with working concentration of Antibiotic
  13. Incubate overnight at 37°C (12-16h)


Colony PCR procedure:
  1. Prepare petri dishes with Ampicillin (50µg/ml) and mark with a pen different areas
  2. Fill a PCR tube with 3µL of water
  3. Pick a colony with a 10µL pipette tip and mark with a pen on the petri dish the colony that you took and give it a number
  4. Put it in the PCR tube with the same number and mix gently
  5. Take the same tip and gently rub one area of the new agar plate to grow new colonies and annotate the associated number on the associated area on the plate
  6. Once every PCR tube have been filled and each area of the plate plated, incubate the plate overnight at 37°C until isolated colonies are seen
  7. Remove the plate from incubator and put it at 4°C.
  8. Put the PCR tubes at 95°C in the thermocycler for 5min for bacterial lysis (to get the plasmids)
  9. Add another total of 7µL (mastermix+primers) into each PCR tube
  10. Run a regular PCR at annealing temperature of 72°C for appropriate elongation time
  11. Load on gel (and remember the associated number of the colony/area of the new plate) and check bands
  12. The clones that give amplicons at correct length are selected for further experiments