Hi Amila,

Yes PEI does dissolve completely. This is what I do:

1.  Stir the PEI in MQ (1mg/ml) and dissolve by heating to ~80 degrees C. Vortex intermittently. It should go completely into solution in a relatively short amount of time.

2. Cool to RT and then pH with 1M Hepes to final concentration of 15mM.

3. Filter through 0.22um filter --> aliquot --> freeze at -20 degrees C. Once thawed a working tube of PEI can be kept at 4 degrees C.

Hope this is helpful.

Good Luck!

Hi Tom, thanks for your answer. I forgot to add in the description that I intend to use the PEI for transfections of cell lines. Do you use it for that as well? I'm not sure I understand your second point 'pH with 1M Hepes to final concentration of 15mM'. What pH does your solution become?

Yes I do use it for transfecting cell lines for transient expression and lentivirus production. Works just as well as other transfection reagents but much less expensive per transfection.

The pH is around 7 after adding the Hepes. I don't think it needs to be exactly pH 7 since you are using such a small amount of it and you are adding it to a larger volume of media which is the optimal pH so that small amount will not significantly change the overall pH.

Hi Tom, I dissolved the PEI at 80C as you suggested. I then adjusted the pH, filtered it, and stored it at -80C. The next day when I thawed an aliquot it was cloudy again and I saw precipitate on the bottom of the tube (see images). Does this happen when you prepare your stock too?  Could you let me know which PEI you buy (catalognumber)?

Hi Amila,

I thawed an aliquot after seeing your question and it is perfectly clear and not cloudy/has precipitates in it. Not sure why that would happen. After I make the stock solution I aliquot 1ml into tubes and freeze at -80 and then just take out a tube when I need it.

I use linear PEI 25,000 MW from Polysciences (Catalog #23966).

Hope this is helpful.

Hi, Amila, 

I have come to the same problem with you. What I met is exactly what your describe.

So I am wondering how did you solve the problem?

Does it have any influence on your transfection?

Hi Guang-Yuan, 

I have tried four ways to dissolve the PEI:

-First one: dissolving 1mg/ml by lowering the pH and then increasing it again to 7, filter, aliquot and freeze. In this case I do not get the precipitation after thawing. 

-Second one: dissolving 0.323mg/ml by heating it to 55C while adjusting the pH to 7. This takes very long and it is necessary to stir days and also overnight @ 55C. After everything is dissolved and pH is stable at 7, filter, aliquot and freeze. In this case I do get precipitation after thawing. 

-Third one: dissolve 1mg/ml by stirring at ~80C. This doesnt take that long and after pH is adjusted to pH 7, filter, aliquot and freeze. In this case I do get precipitation after thawing. 

-Fourth one: dissolve 1mg/ml by microwaving for 30" @ 700W. This is the quickest method. Adjust pH to 7, filter, aliquot and freeze. In this case I do get precipitation after thawing. 

So I guess heating it in any way causes precipitation after thawing. In any case, HEK293 transfection using PEI made by method 1 works. I have also tried PEI transfection made by method 4 which also works, even though there is precipitation, and I didnt see a transfection difference between the PEI from the two methods. I didnt test the PEI made by methods 2-3. However, for my experiments I'm using the PEI from method 1, simply because I didnt see any precipitation. 

Strange thing is, methods 2-4 were performed previously by other researchers (and I guess with other LOTs) and they did not report any precipitation after thawing. Have you purchased the PEI recently? Perhaps its a strange LOT.. 

hope it helps...

Hi Amila

What reagent do you use for adjusting pH of the solution?

In our lab, we use PEI from Sigma (solution) 50% W/V. so you dont need worry about dissolving PEI anymore.

PEI from sigma is so viscous, how do use it. Unable to take the consistent volume.

Hi @Viji N, you can use w/v measurement. In our lab we normally use the 100 ul or 1 ml pipette tips to take PEI sample and drop into your weighing bottle. Just dip your pipette tip in the PEI and roll like a gel around the tip. v/v measurement does not work because PEI 50% (sigma) is highly viscous.

Okay that's working. Thank you Ms.Vincentia.

Dear Amila

You should heat ddH2O to 80C then add the pei and mix by vortex briefly.

Best regards

See below

Good luck

Dear Zuko,

I'm wondering if it is possible to use H2So4 for getting pH of less than 2 instead of using HCl as suggested by many literature for preparing PEI. Kindly advice.

Hello all.

Anybody can tell how to handle this PEI polymer. I am using branched polymer and it is very viscous, waxy texture, very difficult to weigh.

Kindly provide your experience with this issue.

Thank you.

First, I don't suggest neutralizing the pH. My lab has been doing that and getting inconsistent results. As a result, they've switched to CaPO4 😖

Take a look at this

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3303003/

Also, I think rapid cooling can cause precipitates. Try cooling in fridge and see if it has a precipitate before freezing.

Guess any of these protocols should be fine.

Best for me is the answer by Ling Zhang, there is nothing like following manufacturer instructions. And, actually, these procedure is the same than that of CSH protocol series (link beneath):

http://cshprotocols.cshlp.org/content/2008/3/pdb.rec11323.full

Easy, efficient ...and very cheap!!!!!

Good luck with your transfections (that could be improved by using endotoxin-free DNA!!! especially for viral production)

Josema

I have prepared 10 ml of 10 mg/ml stock of PEI. 100 mg was dissolved in 10 ml of water and completely dissolved after adjusting the pH as per recommendation Nicole Lau

https://www.polysciences.com/skin/frontend/default/polysciences/pdf/23966_proc1.pdf

Hi everyone,

I prepared 1 mg/mL PEI (Polyethylenimine, linear average Mn 5,000, PDI ≤1.3, Sigma) using the heating process. I dissolve 5 mg PEI in 5 mL ultrapure water and vortex, then I heat at 85 centigrade and it completely dissolved.

I followed this paper: Article Transient Mammalian Cell Transfection with Polyethylenimine (PEI)

10mg to 9.5ml millipore water, shake in 37C for 10min or so. Completely dissolved. Should be clear. Then add slowly around 50-100ul 1N NaOH. Make sure measure pH and keep the pH below 7. Once reached around 6.9. Can stop adding NaOH. Never over-titrate. I tried and it turned cloudy (like precipitation). Still clear in pH 7.0!