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Help with concentrating lentivirus

lentivirus peg 6000 centrifuge cloning transfection

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#1 Davegatc

Davegatc

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Posted 09 July 2015 - 08:27 PM

Hello everyone,

 

I recently created an Expression vector using invitrogen's "Block-It HiPerform lentiviral Pol II miR RNAi Expression system with EmGFP" protocol (https://tools.lifete...iR_RNAi_man.pdf). 

 

I ran a transfection with the expression vector along with the viral packaging plasmids to produce lentivirus in the 293FT cell line. Everything worked as planned and the 293 FT cells express GFP upon transfection with the plasmids (this step is detailed on pages 37-43 of the attached manual).

 

I am transfecting the 293FT cells in a T75 flask and I use 13 mL of serum-free medium to harvest the lentiviral supernatant (the serum free medium is added around 16 hours after the transfection and I add L-glutamine/sodium pyruvate/NEAA/pen-strep to the media ).

 

The problem comes when I try and concentrate the virus.

 

Since I am limited in the instruments that I can use (I cannot use a ultracentrifuge at the moment), I recently tried using PEG 6000 to concentrate the lentivirus using a protocol suggested in Nature

 

http://www.nature.co...ot.2009.22.html

 

This is the protocol that I adapted from natures recommendations:

  1. Take the 13 mLs of the filtered viral supernatant from the T75 flask and add it to a 50 mL conical
  2. Add 3.25 mL of PEG 6000 (50% stock) to the conical
  3. Add 1.338 mL of 4M NaCl stock to the conical
  4. Add 1.485 mL of PBS (without CaCl or MgCl) to the conical
  5. aliquot the solution from the 50 ml conical into 13x eppendorfs 
  6. Store the eppendorfs at 4 degrees for 1.5 hours. Mix contents every 20-30 minutes
  7. Centrifuge the eppendorfs at 7,000g for 10 minutes at 4 degrees. A white pellet should form
  8. Carefully decant the supernatant and add 50 µl of cell media to each Eppendorf
  9. Resuspend the pellets by vigorously pipetting the liquid up and down 
  10. Vortex the eppendorfs Vigorously for 20-30 seconds
  11. Transfer the vector suspension into screw-cap microfuge tubes in aliquots of 50 µl
  12. Snap-Freeze the tubes in crushed dry ice and store them at -80 degrees

 

The reason why I aliquoted the solution into 13x eppendorfs is because the only centrifuge that I have which reaches 7000g  takes only 1.5 mL eppendorfs.

When I tried this protocol, there was no pellet after running the sample at 7000g for 10 minutes (step 7) and I wasn’t able to get any lentivirus.

 

So now I am in a bind...

 

Is there something that I might be missing with the Peg 6000 protocol?

 

As a possible back up option.... The centrifuge that I use with the eppendorfs can reach 20,800g. Can I concentrate the lentivirus with the 20,800g centrifuge if I run it for long enough at 4 degrees and then remove the top portion of the supernatant?

Thanks for the help!!!!


Edited by Davegatc, 09 July 2015 - 10:51 PM.






Also tagged with one or more of these keywords: lentivirus, peg 6000, centrifuge, cloning, transfection

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