SOP for Semi-Dry Electro Blotting Unit
1.0 OBJECTIVE
To establish a Standard Operating Procedure (SOP) outlining
the operation, maintenance, and calibration of the Semi-Dry Electro Blotting
Unit.
2.0 SCOPE
This SOP applies to the operation, maintenance, and
calibration of the CBS Scientific Semi-Dry Blotting System for
biochemical and molecular biology studies.
3.0 RESPONSIBILITY
3.1 All personnel in the Biochemistry Department are
responsible for implementing and adhering to this SOP.
3.2 The Head or designees of the Biochemistry Department are
responsible for ensuring compliance with this SOP.
4.0 DISTRIBUTION
4.1 The Quality Assurance department maintains the SOP ‘Master Copy’ approved by the Quality Manager.
4.2 Control copies of all SOPs are distributed to user
departments and placed near relevant equipment as display copies.
5.0 DEFINITIONS & ABBREVIATIONS
5.1 Definitions
5.1.1 **Blotter:** A method in molecular biology and genetics
to transfer proteins, DNA, or RNA onto a carrier (e.g., nitrocellulose, PVDF,
or nylon membrane) after gel electrophoresis or directly onto the membrane.
Visualization is achieved through various staining or labeling techniques.
5.2 Abbreviations
5.2.1 **SOP:** Standard Operating Procedure
5.2.2 **BCM:** Biochemistry
5.2.3 **PVDF:** Polyvinylidene fluoride
5.2.4 **DNA:** Deoxyribonucleic acid
5.2.5 **RNA:** Ribonucleic acid
5.2.6 **mA:** Milliampere
6.0 PROCEDURE
6.1 Principle
The semi-dry blotting system facilitates reliable and quick
transfer of western, northern, or southern blots, accommodating gel dimensions
up to 12 x 34 cm. It employs durable and corrosion-resistant stainless steel
and platinum-coated titanium electrodes to create a uniform electric field for
effective transfer onto various substrates.
6.2 Operation
6.2.1 General Operation
6.2.1.1 Transfer efficiency depends on multiple factors
(e.g., gel concentration, thickness, molecule size, shape, net charge), so
results may vary.
6.2.1.2 For resolving gels, an acrylamide concentration
range of 8-10% separates proteins efficiently (14-66 kD) for transfer.
6.2.1.3 Apply a maximum current of 0.8 mA/cm² of gel area
for electrophoretic transfer.
6.2.1.4 Ensure an adequate power supply.
6.2.1.5 Typical runs take 15-60 minutes, depending on gel
size and type of transfer.
6.2.2 Working Procedure
6.2.2.1 Cut 2-4 pieces of blotting paper (0.35 mm thick) and
a membrane piece to the gel's size. Avoid touching the membrane with bare
fingers.
6.2.2.2 Pre-equilibrate the membrane in buffer if required.
For PVDF membranes, activate in 100% methanol for 30 seconds and drain excess
liquid.
6.2.2.3 Soak 2-4 pieces of blotting paper in transfer
buffer, ensuring at least 2 mm overlap on each side.
6.2.2.4 Place six pre-soaked filter paper pads on the base
electrode plate (anode).
6.2.2.5 Place the membrane on top of the filter pads,
ensuring no air pockets.
6.2.2.6 Place the gel on the membrane and smooth out air
pockets.
6.2.2.7 Place six more filter paper pads on the gel and
smooth out.
6.2.2.8 Ensure proper alignment of blotting papers,
membrane, and gel.
6.2.2.9 Roll with a glass rod to remove residual air
bubbles.
6.2.2.10 Secure the lid using screws tightened evenly.
6.3 Running the Blot
6.3.1 Connect the leads (red to positive base, black to
negative lid).
6.3.2 Attach power leads to corresponding sockets on the power supply.
6.3.3 Set the blotter at 0.8 mA/cm² of gel.
6.3.4 Adjust run time based on molecule size. Generally,
larger proteins and nucleic acids require 2 hours, while smaller molecules need
less time.
7.0 PRECAUTIONS
7.1 Do not autoclave or dry-heat sterilize components.
7.2 Avoid exposure to phenol, acetone, benzene, halogenated
hydrocarbons, or alcohols.
7.3 Avoid prolonged UV light exposure.
7.4 Do not treat with DEPC-treated water for extended
periods at 37°C.
7.5 Handle platinum-coated titanium electrodes carefully to
avoid damage.
7.6 Ensure correct polarity to prevent electrode damage.
7.7 Ensure proper power connections before initiating
transfer.
7.8 Clean components with water and non-abrasive detergent,
avoiding abrasive cleaners and scouring pads.
8.0 REFERENCES & FORMATS
8.1 References
8.1.1 [CBS Scientific Semi-Dry Blotting
Systems](https://ca.vwr.com/store/product/en/4635236/semi-dry-blotting-systems-cbs-scientific)
8.1.2 [VWR Semi-Dry Blotting
Systems](https://pr.vwr.com/assetsvc/asset/en_US/id/12998433/contents)
8.2 Formats
- Format I: Log Book Maintenance
- Format II: Document History Sheet
END OF DOCUMENT
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