Editing Lentivirus Concentration PEG

1. Harvest the cell supernatant containing lentiviral vectors

Collect the supernatant in 50 ml VWR tubes 2 days and 4 days after transfection. Combine and spin at 3000 rpm for 10 min at 25 <sup>o</sup>C to pellet cells and large cell debris.



2. Collect supernatant, alternatively, filter the supernatant using a 0.45-mM PES filter.

3. New protocol: add 5X PEG, for example, add 50 ml 5X PEG solution to 200 ml medium.

'''5XPEG:'''

1000 L

500g PEG 6000 ( final is 50% )

Add 88 g solid NaCl&nbsp; (final is 1.5 M NaCl)

Add water to 1 L.

Autoclave.



'''PEG/NaCl (5x) stock solution'''

PEG-8000 20%, NaCl 2.5 M. Dissolve 100 g PEG-8000 (20% w/v) and 75 g NaCl (2.5 M) in 400 ml ddH20 and bring to a final volume of 500 ml by stirring at RT. The solution can be autoclaved (optional) but mixing during the cooling period is required to prevent a phase separation. Store at RT.

Final Conc: 4% PEG-8000. 0.5 M NaCl.

The following is old

&nbsp;&nbsp;&nbsp; '''For 204 ml supernatant''':

&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Add 51 ml of a 50% PEG 6000 solution to 204 ml lentivirus-containing supernatant,

&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; then add add 21.7 ml of a 4 M NaCl stock solution.<br/>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; add 23.3 ml of PBS. This will result in a final volume of 300 ml.

&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; The final Concentration: PEG 6000: 8.5% NaCl: 0.3 M.

&nbsp;&nbsp;&nbsp; '''For 35~38 ml lentivirus-containing supernatant in 50 ml tubes'''

&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; add 7~8.5 ml 50% PEG 6000 and 4~5&nbsp; ml 4 M NaCl.

&nbsp; &nbsp; '''For 150 ml supernatant from three T175 flasks''' (25 ml x 2 collections x 3 flasks=150 ml)

&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Add 35 ml of a 50% PEG 6000 solution to 204 ml lentivirus-containing supernatant,

&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; then add add 15 ml of a 4 M NaCl stock solution.<br/>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; This will result in a final volume of 200 ml;&nbsp; PEG 6000: 8.75% NaCl: 0.3 M

<br/>4. Distribute the sample as 150-ml aliquots in two 250-ml polypropylene wide-mouthed bottles (or VWR 50 ml tube).

5. Store the bottles at 4 oC for 1.5 h. Mix contents every 20~30 min.

6. Centrifuge at <strike>7,000g</strike> 6000rpm for 15 min at 4 <sup>o</sup>C.

7. After centrifugation, a white pellet should be visible.

8. Carefully decant the supernatant and add <strike>1ml</strike> 5 ml autoclaved PBS (or other buffer) to the centrifuge tubes.

9. Resuspend the pellets by pipetting liquid gently up and down.

10. Transfer suspension to 100KDa spin concentrator.

11. Add 2 X 5 ml of autoclaved PBS to wash centrifuge tubes, transfer liquid to 100kDa spin concentrator.

12. Spin at 3000 rpm for 15 min to 1 hour.

13. Add 10 ml autoclaved PBS to spin concentrator, spin for another 15 min to 1 hour.

14. Transfer remaining liquid into screw-cap microfuge tubes in aliquots of 200 ul.

15. Add&nbsp; 1 ml autoclaved PBS to wash the spin concentrator. Do not aliquot this wash.

16. Titer virus by RT-PCR, or 293 cell infection or p24 ELISA.

17. Print labels (Catalog number, Product/virus name, Lot #, Date, Titer, Company Name).

18. Snap-freeze the tubes in crushed dry ice and store at -80 <sup>o</sup>C.



'''4.0 M NaCl''' stock solution

Sterilize by autoclaving. This solution can be stored at 4 <sup>o</sup>C for at least 1 year.

'''50% PEG 6000'''

Dissolve 250 g of PEG 6000 in 500 ml of H2O. Autoclave and mix thoroughly. This solution can be stored at 4 <sup>o</sup>C for at least 6 months.
[[Category:Protocols]]