Environmental considerations,

➢ AIR HANDLING
- So in air handling, for extremely high performance PCR laboratories that will be involved
with detecting very low prevalence DNA or RNA molecules. When we say very low
prevalence, not common, such as the infectious disease agents that is detected in clinical
samples.
- And so, the additional measures may be necessary to prevent contamination from the air
being recirculated between the pre and post PCR laboratories. So there's always a
contamination from the air that might enter the laboratory. So in this case, the air handlers
need to be separate and the air pressure individually adjusted in each laboratory.

Pre-PCR laboratory,

- there should be a slight positive pressure compared to the air that is connecting to the
corridor or the hallway. It shouldn't go out or it prevent that the air to go outside the area.
- And it also have insulation, it prevents the air from escaping and also entering in the room.
- As you can see in the image, there is a two plus sign, which means slightly positive.
- This is because during pre-PCR, we are going to prepare all the requirements and protocols
for isolation of nucleic acids.
- - we do not want to contaminate them with other infectious diseases. That might change
their structures and genomic sequences.

Post-PCR laboratory,

- In contrast, it should be at slightly reduced pressure para mag-pull siya ng air from the outside.

- For example, this is the air. It will pull the air from the outside. And thereby, it will prevent the escape
of amplicons from the complicated PCR samples being analyzed inside the lab.

- AMPLICONS is a segment of either DNA or RNA.

➢ So finally, the air handlers for the pre- and post-PCR laboratories need to be connected to separate
air ducts and each must lead to a separate location for exhaust.

➢ UV IRRADIATION .
- It is possible to exploit further the sensitivity of nucleic acid to ultraviolet rays by using UV
to sterilize the entire pre-PCR laboratory.
- ultraviolet rays is use to sterilize the PCR laboratory. So this can be done by having UV lights
placed in the ceiling fixtures and connecting their activation to a lockout mechanism on the
exit door so they only illuminate when the last person in the lab closes and locks the
external lab door.
 ❖ UV rays in the biosafety cabinet.
- This type of hardware is installed, it must be accompanied by a ventilation system to
eliminate the UV-generated ozone.
- They are scheduled for monitoring the performance of the UV bulbs.

Protective clothing.

- to further prevent PCR applicants from leaving the post-PCR laboratory.

- And each investigator or laboratory staff should have a dedicated post-PCR laboratory coat.

- each investigator should have a general molecular biology laboratory coat. And a separate coat for pre-
PCR.

- they should have a disposable gown or boots na susuotin nila.

- its purpose is to prevent cross-contamination.

Adhesive paper at lab entrances

- this approach effectively prevents trace amounts of dust and debris from entering laboratory.

- It is a rather expensive approach to control contamination. But this is worth the expense for selected
applications which we can prevent cross-contamination.

environmental considerations, its purpose is to prevent cross-contamination when we are performing

with the different methods that might affect.

Sterilization of reagents.

- So because PCR laboratories perform some molecular biology methods that requires sterile
reagents, some may need to be autoclaved.
• Autoclave - it kills any kind of microorganisms,
- they are used to sterilize equipments para mapatay yung mga other
microorganisms that was used or that might be contaminated.

- So the single most critical reagent is water. The sterile USP water.

✓ USP - United States pharmacopoeia.

- Meaning that kung ano yung requirement or the standard of the water na dapat daw sterile
siya.
 - Yung ano yung magiging standard niya or yung requirement para matawag mo siyang sterile
water.
✓ So this can be quickly converted to PCR water by filtering it through 0.45 micron or through two
0.45 micron nitrocellulose filters.
- So these filters have a very high binding capacity for nucleic acid and proteins.
✓ high binding - the filter, can prevent the protein or the nucleic acid to contaminate the other
reagents. That's why we also have the nitrocellulose filters.
- So if the laboratory is involved in amplification of very small quantities of bacterial DNA,
yung USP water should be autoclaved separately from all other reagents before filtration.
❖ the reagents and solid items destined for the pre-PCR lab should be autoclaved separately from
other supplies.
❖ And it is also important to know that spent tissue culture fluids, bacterial media plates with
recombinant cultures or plasmid, and samples from the post-PCR laboratory, they represent a
large potential reservoir of contaminating DNA.

➢ Bacteria, cultures,plasmids, - will contaminate the DNA and should also be autoclaved
separately from any material that will enter the pre-PCR laboratory.