Aug 16, 2024
Titration of Lentivirus
Forked from a private protocol
- 1Lund University
Protocol Citation: Anita Adami 2024. Titration of Lentivirus. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn6yj6l5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 16, 2024
Last Modified: August 16, 2024
Protocol Integer ID: 105751
Keywords: Titration, lentivirus, DNA isolation, qPCR, titer, ASAPCRN, titrating lentivirus, titration of lentivirus, lentivirus, titration, protocol
Abstract
This protocol is about titrating lentivirus.
Attachments
Materials
qPCR [Taqman]
Standard qPCR protocols were then performed using the following primers:
a. Woodchuck hepatitis virus post-regulatory element [WPRE]:
WPRE FP- GGCACTGACAATTCCGTGGT,
WPRE RP-AGGGACGTAGCAGAAGGACG
WPRE probe- 5´Fam-ACGTCCTTTCCATGGCTGCTCGC -Tamra-3´.
b. Human albumin was used as a standard:
ALB FP-5´-TGAAACATACGTTCCCAAAGAGTTT-3´
ALB RP 5´-CTCTCCTTCTCAGAAAGTGTGCATAT-3´
ALB probe- 5´Fam-TGCTGAAACATTCACCTTCCATGCAGA-Tamra-3´.
c. LV 2 for LV not using WPRE:
LV2 FP; 5´-ACTTGAAAGCGAAAGGGAAAC-3´
LV2RP; 5´CACCCATCTCTCTCCTTCTAGCC-3´
LV2 PROBE ; 5´.FAM AGCTCTCTCGACGCAGGACTCGGC-TAMRA-3´
Relative quantification of WPRE/LV2 and albumin content compared to reference batch [virus of known titre] was performed by the ΔΔCT method.
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Virus transduction
3d
When aliqouting the virus, prepare 1 vial with 6 μl for titration. This vial must go through one cycle of freeze / thaw before titration and if you want to titer in conjunction with aliqouting you can put the vials on dry-ice for a bit.
Seed cells in 6 well plates seed 100,000 cells - in 2 mL - 3 mL media . For each virus - 3x wells are required. Plate 6x additional wells for control and reference batch.
Dilute the virus 1:10 before adding it to the cells [54 µL PBS + 6 µL virus ].
Then add 3 μl, 10 μl and 30 μl of the diluted virus to the cells [corresponding to 0.3 μl, 1 μl and 3 μl undiluted virus].
Leave for 72:00:00 before DNA is isolated.
3d
Isolation of DNA
10m
Look at the cells under the microscope and compare with control.
Remove media and wash 1x with 1 mL PBS [Use 1000 ul pipette and don´t add the PBS directly on the cells to avoid flushing them off].
Add 500 µL trypsin and leave for a few minutes in the incubator.
After incubation with trypsin, add 500 µL PBS or Media and transfer cells to 1.5 ml tubes.
Spin down at 300 x g, 00:05:00 .
Remove supernatant with a pipette to not disturb the pellet.
Make a master mix of 200 µL PBS + 20 µL Protein K per sample and resuspend the pellet in 220 µL mastermix .
Add 200 µL Buffer AL [w/o ethanol] and mix thoroughly by vortexing.
Bring samples to DNA isolation room and follow the instructions from the Qiagen DNeasy Protocol: Purification of Total DNA from Animal Blood or Cells [Spin-Column Protocol]. Start with incubation at 56 °C for 00:10:00 and then move directly to step 3.
10m
Assess titre using qPCR.