TITLE: Optimized Cloning Protocol for Standard Curve Generation in qPCR of Environmental Functional Genes
AUTHORS: Abul Bashar, Raju Podduturi, Amaru Miranda Djurhuus, H. M. Rakibul Islam, Mohammad Mahfujul Haque, Lars Hestbjerg Hansen, Niels O. G. Jørgensen

[DESCRIPTION]
Due to the complex and varied biochemical requirements and physiological behaviors of certain environmental bacterial and archaeal groups that carry specific functional  genes as potential biomarkers, it is challenging to extract and maintain pure cultures of these organisms. Therefore, generating standard curves for qPCR necessitates cloning gene sequences from environmental DNA samples.
This protocol was developed for cloning target genes from environmental samples to produce plasmid DNA, which was then used to prepare standard curves for quantifying functional genes, including mcrA, pmoA, amoA, and nosZ from water and soil samples. The procedure involves inserting amplified gene sequences into pGEM‱-T Vectors and transforming them into JM104 E. coli competent cells. Testing this protocol yielded higher amounts of pure plasmid DNA, making it suitable for standard curve preparation for qPCR of the target genes.

[STEPS]
SECTION: Step-1: Amplification of Target Gene Sequence
1. Amplify the target gene sequences from environmental DNA using a PCR reaction. The reaction mixture should include polymerase (0.5 μL), buffer (5 μL), dNTPs (5 μL), forward primer (1 μL), reverse primer (1 μL), template DNA (3 μL), and water (32.5 μL).
  
Note: The presence of humic acid or other contaminants may impact the efficiency of the PCR reaction. Therefore, it is recommended to assess the purity of DNA samples using a Nanodrop spectrophotometer.
SECTION: Step-1: Amplification of Target Gene Sequence
2. Thermal condition: 
1Initial Denaturation: 95 ⁰C   3-5 min.
               Denature:  95 ⁰C  30sec.
2Cycles  3Anneal: 55-60 ⁰C  30 sec. 
               Extend: 72 ⁰C 30 sec.
Extension: 72 ⁰C  5 min.
Hold: 4 ⁰C  Indefinitely

1Initial Denaturation: The temperature and duration of the initial denaturation step depend on the sample type. DNA extracted from soil may require a longer denaturation time due to potential chemical contamination.
2PCR Cycles: Perform 30 cycles for the mcrA gene, 35 cycles for the pmoA and nosZ genes, and 40 cycles for the amoA gene.
3Annealing Temperature: Set the annealing temperature to 55°C for mcrA and nosZ, 60°C for pmoA, and 57°C for amoA.
SECTION: Step-1: Amplification of Target Gene Sequence
3.  Primer dimers and other PCR components often form during the reaction and should be removed. This can be done using DNA Clean and Concentrator kits according to the manufacturer’s instructions or by using AMPure XP beads.

 Note: When using beads for cleanup, add 9 µL of beads to a 15 µL sample. After washing with ethanol, ensure all ethanol is completely removed by pipetting and allowing the sample to air dry.
SECTION: Step 2: Ligation to Vector
4. Thaw the ligation buffer, vector (pGCM T vector), PCR products, and T4 DNA ligase on ice.
SECTION: Step 2: Ligation to Vector
5. In a 1.5 mL Eppendorf tube, combine 2.5 μL of ligation buffer, 0.5 μL of vector (pGCM T vector), 2.0 μL of cleaned PCR products, and 0.5 μL of T4 DNA ligase. Gently mix the reaction and incubate at room temperature (22–23°C) for 5 minutes. It is recommended to include a negative control to check for uncut plasmid contamination.

Note: The cloning reaction can be stored at −20°C until further use.
SECTION: Step-3: Transformation
6. After removing the competent cells (JM104: E. coli) from the freezer (-80°C), place them on ice for 20 minutes. Mix 3 μL of the cloning mixture (including the negative control) with 50 μL of competent cells in a 1.5 mL tube.
SECTION: Step-3: Transformation
7. For heat shock, keep the reaction on ice for 30 minutes, then transfer it to a 42°C water bath for 45 seconds. Immediately transfer the tube back to ice for 2 minutes.

Note: To optimize the transformation efficiency, it is crucial to maintain precise temperature and timing. It is recommended to set the water bath to 42°C before starting the heat shock to avoid any delays.
SECTION: Step-3: Transformation
8. Add 300 μL of LB media (without antibiotics) or SOC media to the reaction and incubate at 37°C for 90 minutes. 

Note: It is highly recommended to place the reaction on a shaker during incubation.
SECTION: Step-4: Culture and isolation of pure transformed cell
9. Prepare plates with solid LB media containing yeast extract (10 g/L), NaCl (5 g/L), tryptone (10 g/L), and bacteriological agar (15 g/L). To inhibit the growth of non-competent cells, add 1 mL of ampicillin (100 mg/μL) to the media. For blue-white colony screening to differentiate between recombinant and non-recombinant colonies, add 4 mL of X-gal (20 mg/μL) and 5 mL of IPTG (100 mM) during media preparation.

Note: The pH of the media should be maintained around 7. Store the plates in the refrigerator until use.
SECTION: Step-4: Culture and isolation of pure transformed cell
10. Label the plates accordingly, then pipette 100 μL and 200 μL of the transformed reactions onto separate plates. Spread the mixture evenly using a sterile spreader. Incubate the plates at 37°C overnight.
SECTION: Step-4: Culture and isolation of pure transformed cell
11. In 50 mL falcon tubes, prepare 10 mL of LB-amp broth containing 5 g/L yeast extract, 10 g/L NaCl, 10 g/L tryptone, and 1 mL/L ampicillin (100 mg/μL). From the blue and white colonies, select pure white colonies (indicating successful transformation) and transfer a single colony from each plate into the LB-amp media. Place the falcon tubes in a shaking incubator at 37°C overnight.

  
Note: LB-amp media can be prepared in advance and stored at room temperature in a sealed condition.
SECTION: Step-5: Plasmid DNA extraction and verification
12. Centrifuge the cultures at 8,000–9,000 rpm for 5 minutes and carefully remove all the supernatant. Use the resulting pellets for plasmid DNA extraction following the QIAprep Spin Miniprep kit manufacturer's instructions. For additional details, refer to the protocol documented by Kodackattumannil et al.1.
SECTION: Step-5: Plasmid DNA extraction and verification
13. Determine the purity and quantity of the plasmid DNA using a NanoDrop spectrophotometer and a Qubit fluorometer, respectively.  

Note: If the quality of the plasmid DNA is suboptimal, consider cleaning it using a DNA Clean and Concentrator kit or beads.
SECTION: Step-5: Plasmid DNA extraction and verification
14. Verify successful transformation through Sanger sequencing and diagnostic restriction digestion followed by gel electrophoresis. For the latter, mix 1 μL of plasmid DNA (300-500 ng/μL), 5 μL of CutSmart buffer, 1 μL of EcoRI HF restriction enzyme, and 43 μL of water. Incubate the mixture at 37°C for 5 minutes. Running the reaction on a 1% agarose gel should yield two distinct bands, representing the vector DNA and the target genes, confirming successful insertion of the target gene sequence into the vector.
SECTION: Step-5: Plasmid DNA extraction and verification
15. Calculate the number of plasmid DNA copies using the following formula:
             Number of copies=(ng×[6.022×1023])/(length ×[1×109]×650)
SECTION: Step-5: Plasmid DNA extraction and verification
16. Store the plasmid DNA at -20 °C for downstream usage including for generating
standard curve.