Weiss Lab:Notebook/Non-Integrating Lentiviral Vectors

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Site-directed Mutagenesis

November, __, 2009 Used QuickChange Multi Site Directed Mutagenesis Kit from Strategene

Three Mutants: 1. IN D64V 2. IN D64V, N120L, Q148A, and W235E 3. IN K264, K266R, K273R

Primers:

November 15, 2009

Transformation into XL10 Competent Cells

Mini-prep

1. Culture isolated colonies from plates in liquid broth over night.

Digest to check fragment sizes

DNA Sequencing

November 23, 2009

1. Dilute primer to 100 uM.

  • oS-LW-INT-RV @ 30.8nmole. Add 308 uL to make 100 uM stock. Dilute to 8 uM (add 1uL of stock to 11.5 uL of sterile water).
  • oS-LW0INT-FW @ 27.5nmole. Add 275 uL to make 100 uM stock. Dilute to 8 uM (add 1 uL of stock to 11.5 uL of sterile water).

2. Add 11 uL of DNA with 1 uL of primer at 8 uM DNA concentrations:

  • Mut 2-1 @ 102.3 ng/uL --> 1125 ng
  • Mut 2-2 @ 40.9 ng/uL --> 449.9 ng
  • Mut 2-3 @ 87.9 ng/uL --> 966.9 ng
  • Mut 3-1 @ 52.6 ng/uL --> 578.6 ng
  • Mut 3-2 @ 241.5 ng/uL (Digest of this did not produce correct fragments)
  • Mut 3-3 @ 104.2 ng/uL --> 1146 ng

3. Send to sequencing center before 4pm! Orders: LW 01: Mut2-1 + FW primer LW 02: Mut2-1 + RV primer etc...

DNA Sequencing Data Analysis

November 26, 2009 (Thanksgiving!)

Results from sequencing:

  • Using reverse primer (oS-LW-INT-RV) gave no sequencing results.
  • Fwd primer (os-LW-INT-FW) gave good alignment results, but sequencing data stopped abruptly after the Integrase gene which is at the end of ORF3. There was no gradual loss of resolution of bases and etc.
  • This leads us to believe that the segment immediately after ORF3 might not exist in this plasmid. This segment is where the reverse primer should have annealed to.

For Mutant 2's, wanted D64V, N120L, Q148A, and W235E. Mut 2-1 had W235E Mut 2-2 had D64V, N120L, and W235E Mut 2-3 had D64V, and W235G

For Mutant 3's, wanted K264, 266, 273R. Mut 3-1 had K264, 266R Mut 3-2 had K266R Mut 3-3 had no mutations.


November 27, 2009

Transformation of Mut 2-2, 3-1, d1-EGFP, d2-EGFP to amplify plasmids.

  • Add 1 uL of plasmid DNA to 20 uL of XL-10 competent cells.
  • Incubate on ice for 40 min.
  • Add 200 uL of SOC media.
  • Plate 20 uL of cells on AmpR agar plates and incubate at 37 C for > 16 hours.

Isolate more colonies from 1st mutagenesis reaction

  • Make 5 culture tubes with 5 mL agar and 1:1000 Amp.
  • Add colonies respectively: Mut 2-4, 2-5, 2-6, Mut 3-4, 3-5
  • Incubate at 37C for > 16 hours.

November 28, 2009

  • Mini-prep 2-4, 2-5, 2-6, 3-4, 3-5. Used 3mL of cell culture, spun down at 13kRPM for 2 min.
Strain Concentration
2-4 95.2 ng/μL
2-5 98.4 ng/μL
2-6 94.7 ng/μL
3-4 389.8 ng/μL
3-5 81.0 ng/μL
  • Observed isolated single colonies for Mut 2-2, 3-1, d1-EGFP, d2-EGFP! =) Will midi-prep on Monday. Put at 4C.

November 30, 2009

Midi-prep of d1-EGFP, d2-EGFP, Mut 2-2 and 3-1.

  • Mut 2-2: 356.3 ng/uL
  • Mut 3-1: 327 ng/uL

December 5, 2009

Virus Packaging:

    1. 148 (WT pol gene): pCMV-dR8.2-dvpr
    • Need 2.8 ug from 750 ng/uL --> 3.73 uL
    1. 149 : pCMV-VSV-G
    • Need 1.4 ug from 1186 ng/uL --> 1.18 uL
  • EGFP
    • Need 1.1 ug from 302 ng/uL --> 3.64 uL
Package pol #149 EGFP
Control 3.73 uL 1.18 uL 3.64 uL
2-2 7.86 uL 1.18 uL 3.64 uL
3-1 8.56 uL 1.18 uL 3.64 uL

Wednesday, December 9 and Saturday, December 12

FACS analysis of infected 293FT cells

  • Prepare a 12-well dish with 1 mL of media
  • Add 200 uL trypsin per well of infected plate, incubate 3 min.
  • Rescue with 1mL media, pipetting up and down well. Add 1000 uL (a little less) to FACS tube, and add remaining cells to the new plate.
  • Keep cells on ice.
  • FACS settings: event count for first day: 2000 (time constraint after solving a lot of machine issues). Event count for second day: 15000.

Wednesday FACS data:

  • .001 file : Mut2-2n2

... etc. Did not save negative control or Mut2-2n1 data.

Tuesday, December 15

Event count is different for all samples (usually around 10000) because it's too slow. Order:

  • Neg control
  • Mut 2-2 n1
  • Mut 2-2 n2
  • Mut 2-2 n3
  • Mut 3-1 n1
  • Mut 3-2 n2 (**lowest: only 5000 events)
  • Cntrl n1 (event count: 8100)
  • Cntrl n2 (event count: 5000)

Observations:

  • Mut 2-2 show a narrower distribution of GFP (between 10^1 and 10^2 of FL1-H, which is the GFP channel. Nice peak curve on the histogram). GFP expression is definitely lower than Mut 3-1 and the Control Integrase package.
  • Mut 3-1 show a wide distribution between 10^1 and 10^4. GFP expression is lower, but probably not as low as Mut 2-2.
  • Control: Distribution between 10^1 and 10^4. Probably multiple viruses affecting one cell. GFP expression is still strong. Low cell count though.
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