PEG for ligation - just filtered by 0.2um filtered (Nov/30/2006 )

Hi, I remember some previous topic talking about ligation with the addition of PEG 8000,

so how is that prepared ? just filtered with 0.2um filter is enough or should it be autoclaved ? The stock solution should be 50% m/v ?

THX

5x Blunt End Ligation Buffer (Invitrogen Ligation Buffer): consists of 250 mM Tris-HCl, pH 7.6, 50 mM MgCl2, 5 mM ATP, 5 mM DTT, 25% (w/v) PEG 8000. To prepare 10 mL of buffer, weigh 2.5 g of PEG 8000 in a 15 mL Falcon 2097 tube that has been treated with antistatic spray or wiped with a sheet of fabric softener. Microwave a 100 mL bottle of Type I (Mili-Q) water for 1 minute on High. Tilt the Falcon tube sideways, spreading out the PEG as much as possible. Add 4.4 mL of hot water and 2.5 mL of room temperature 1 M Tris-HCl, pH 7.6 to the PEG and immediately mix to dissolve. Cool the mix to room temperature and add 500 µL of 1 M MgCl2, 500 µL of 100 mM ATP and 50 µL of 1 M DTT to a final volume of 10 mL. Aliquot for storage at -20°C. Failure to heat the water will result in PEG that will take approximately 24 hours to dissolve.

You can prepare a 40% PEG solution the same way.

I have never bothered to sterillize either mixture, since I prepare it with sterile liquids and store the ligation buffer or 40% PEG in 1 mL aliquots at -20C.

Thank you very much

Indeed, I found that 40% PEG8000 is very viscous, would it result in pipetting error ?

The density of 40% PEG is reported as 1.075 g/mL ("Practical Molecular Biology" http://molbiol.edu.ru). Add this component by weight rather than volume.

This website does not include the molecular weight of the PEG, and the actual monecular weights of each lot of PEG will vary significantly. Use the density value with these caveats in mind.