been adapted from Cold Spring Harbor Protocols [1]. Reagents: 1. 12 M Hydrochloric Acid (HCl). 2. 10 M Sodium Hydroxide (NaOH). 3. Linear Polyethylenimine ...
Protocols for
23966 - Linear Polyethylenimine 25,000 PEI Solution - Acid Method (Recommended) For transient transfection, PEI is typically used at a concentration of 1 mg / mL. This protocol is relatively quick and produces a clear solution that can be used for thousands of transfections. It has been adapted from Cold Spring Harbor Protocols [1]. Reagents: 1. 12 M Hydrochloric Acid (HCl) 2. 10 M Sodium Hydroxide (NaOH) 3. Linear Polyethylenimine (PEI) 25,000 Powder (Polysciences 23966) 4. Milli-Q® water, Water for Injection (WFI), or similar high-purity biological grade water Equipment: 1. 500 to 2000 mL beaker (for 500 mg to 2 g of PEI powder, respectively) 2. 500 to 2000 mL glass graduated cylinder 3. pH meter 4. 2 x plastic pipettes (for pH titrations) 5. Stir Plate 6. Stir Bar 7. 0.22 µm vacuum filter setup 8. Plastic aliquot tubes Method: 1. Weigh 500 mg to 2 g of PEI powder 2. Suspend PEI in beaker with 450 mL to 1.8 L water (i.e. concentration of about 1.11 mg / mL) 3. Add Hydrochloric Acid dropwise until pH < 2.0 ( ~0.8 to 3.2 mL) 4. Cover top of container and stir for 3 hours, or until solution is mostly clear. 5. Add sodium hydroxide dropwise until pH ~ 7.0 ( ~0.5 to 2.0 mL) 6. Transfer to graduated cylinder 7. Add water to adjust final concentration to 1.0 mg / mL 8. Sterile-filter through 0.22 µm membrane 9. Aliquot to desired volumes 10. Store aliquots at -20°C 9/12/2016 Rev 1.1
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PEI Solution - Heat Method While this method is relatively quick and successfully used by many researchers, it may produce a cloudy solution following a freeze-thaw cycle. Reagents: 1. Linear Polyethylenimine (PEI) 25,000 Powder (Polysciences 23966) 2. Milli-Q® water, Water for Injection (WFI), or similar high-purity biological grade water 3. 12 M Hydrochloric Acid (HCl) Equipment: 1. 500 to 2000 mL beaker (for 500 mg to 2 g of PEI powder, respectively) 2. 500 to 2000 mL graduated cylinder 3. Plastic pipette 4. pH meter 5. Hot Stir plate 6. Stir bar 7. 0.22 µm vacuum filter setup 8. Plastic aliquot tubes Method: 1. Weigh 500 mg to 2 g of PEI powder 2. Suspend PEI in beaker with 500 mL to 2 L of water (to final concentration of 1 mg/mL) 3. Cover top of container and heat to 80°C and stir for three hours, or until solution is mostly clear 4. Cool to room temperature 5. Adjust pH to 7 6. Sterile-filter through 0.22 µm membrane 7. Aliquot to desired volumes 8. Store aliquots at -20°C
Transfection Protocol At present we cannot evaluate or prepare protocols for transfection. However, one of the most commonly-cited articles that uses our PEI powder for transfection is publicly available [2].
Storage Unopened powder has an indefinite shelf life at room temperature. Once the container is opened, the powder has a shelf life of two years at room temperature. Frozen aliquots of 1 mg/mL PEI have a shelf life of about 1 year at -20°C. We recommend aliquoting such that the solution does not have to go through more than 1 freeze-thaw cycle.
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References 1. Polyethylenimine (PEI), linear (1 mg/mL). Cold Spring Harbor Protocols 2008, pdb.rec11323–pdb.rec11323 (2008). doi:10.1101/pdb.rec11323 2. Durocher, Y., Perret, S. & Kamen, A. High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Research 30, 9e–9 (2002). doi:10.1093/nar/30.2.e9
