Advanced PCR and In-Fusion Cloning Laboratory Protocols

Reagent Proportions and Volumes:
- The entry vector volume is maintained at $3\,\text{microliters}$ .
- The PCR insert volume is adjusted to $0.75\,\text{microliters}$ .
- The In-Fusion mix is added at a volume of $1\,\text{microliter}$ .
- The reaction is described as a $5 \times$ reaction.
- Nuclease-free water is used to make up the remaining volume to reach a total reaction volume of $5\,\text{microliters}$ .
- Two distinct reactions are prepared: one for binary entry vector plus insert and one for the entry vector alone (serving as a control).

Comparison of LB and SOC Media:
- While LB (Lysogeny Broth) media is frequently used, SOC media is preferred for this procedure.
- SOC is described as a "richer" and more nutrient-rich media.
- The primary purpose of using SOC is to further encourage the bacteria to be "happy" and more receptive to taking in the plasmid.
- SOC media often comes specifically with the Takara kit.
- If SOC media is unavailable, LB media is an acceptable substitute, though SOC is preferred for ligations.
Storage and Handling:
- SOC media should be thawed before use.
- It should be brought to room temperature or warmed to $37\,^{\circ}\text{C}$ .
- Unused sterile SOC media should be marked with an "X" on the tube and stored back in the box for future use.

Step-by-Step Thermal Treatment:
1. Place the reaction on ice.
2. Apply heat (heat shock).
3. Place back on ice.
4. Add the recovery media (SOC or LB).
Incubation Parameters:
- Incubate the mixture at $37\,^{\circ}\text{C}$ for exactly one hour.
Tube Selection and Mixing Optimization:
- Standard $1.5\,\text{ml}$ microfuge tubes have a bevel at the bottom which can hinder efficient mixing.
- The use of $2\,\text{ml}$ round-bottom tubes is recommended specifically for bacterial work.
- The round bottom allows for superior mixing, which is critical when dealing with "low occurring events."
- During incubation, tubes should be placed on their side to ensure maximum mixing across the surface area.

Initial Plating:
- Extract $150\,\text{microliters}$ of the transformation culture and plate it directly.
Concentrated Plating (The "Dump Plate"):
- The remaining volume (the remainder of the culture) is centrifuged to form a pellet.
- The supernatant is removed and discarded.
- The bacterial pellet is resuspended in approximately $100\,\text{microliters}$ of fresh SOC media.
- This concentrated suspension is plated entirely on a separate plate, referred to as the "dump plate."
Volume Tracking:
- Each transformation requires $500\,\text{microliters}$ initially plus approximately $100\,\text{microliters}$ for resuspension.
- For two samples (vector+insert and vector alone), the total SOC requirement is approximately $1,200$ - $1,400\,\text{microliters}$ .
Handling Precautions:
- Competent bacteria have weakened cell walls and must be handled with care.
- After one hour of growth in SOC, the bacteria should have undergone a couple of rounds of replication and rebuilt much of their cell walls.

The "Xerox Machine of Biology":
- Bacteria act as a biological Xerox machine, making many copies of the circular plasmid.
- In-Fusion cloning relies on homologous recombination.
- Successful cloning requires a true closed circular plasmid.
Kanamycin Selection:
- The plasmid contains a kanamycin resistance gene.
- When the DNA is transcribed into RNA and then translated into the kanamycin resistance protein, the protein is "spit out" by the bacteria.
- This allows only the bacteria containing the closed plasmid to grow and form colonies on kanamycin-treated plates.
In-Fusion vs. Traditional/Restriction Cloning:
- Traditional cloning (restriction cloning) involves the use of DNA ligase, which works effectively on both single-cut and double-cut vectors.
- In-Fusion cloning typically has a lower background because the homologous recombination process does not favor the closing of unmatched ends as effectively as a ligase might.

Negative Control (Entry Vector Alone):
- This control is intended to show no colony growth if the vector was properly cut.
- If colonies appear on the negative control plate, it may indicate:
  1. Incomplete digestion: The plasmid remained uncut and retained its original form (e.g., original EGFP).
  2. Single-cut event: Only one restriction site was cleaved, allowing the linear DNA to potentially close back up.
  3. Background: Contamination or low-efficiency ligation of the vector itself.
Analyzing Results:
- High colony counts on the experimental plate versus low counts on the control plate indicate high efficiency.
- If both plates show high colony counts, the "screen number" must be increased because of high background noise.

Question: Why is the vector-only plate considered a negative control?
Answer: Because it does not contain the FGF2 insert. If the vector is double-cut (using enzymes identified in the transcript as "Eczema, iron, and sarcophagus"), it should not be able to recircularize and confer kanamycin resistance without the insert provided by the In-Fusion reaction.
Question: What if both the experimental plate and the control plate show colonies?
Answer: That indicates that both the FGF2 and the EGFP (original vector) might be present. This requires a higher number of colonies to be screened to differentiate the successful clones from the background.
Question: Should this part of the experiment be done immediately?
Answer: No, the transformation and plating are involved processes and can be deferred until the following day.
Question: Is it necessary to use a specific tube type?
Answer: Yes, $2\,\text{ml}$ round-bottom tubes are preferred over $1.5\,\text{ml}$ tubes to ensure better mixing, especially for low-probability ligation events.