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pRosetta
(Plasmid #59700)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 59700 Plasmid sent as bacteria in agar stab 1 $65
Lentiviral Prep 59700-LV Virus (1 mL at titer > 1x10⁶ TU/mL)
and Plasmid. More Information
$250

This material is available to academics and nonprofits only.

Backbone

  • Vector backbone
    pLKO.1
  • Total vector size (bp) 8765
  • Vector type
    Mammalian Expression, Lentiviral
  • Selectable markers
    Puromycin, Blasticidin

Growth in Bacteria

  • Bacterial Resistance(s)
    Ampicillin
  • Growth Temperature
    37°C
  • Growth Strain(s)
    Stbl3
  • Copy number
    High Copy

Gene/Insert 1

  • Gene/Insert name
    Puro
  • Tag / Fusion Protein
    • 2A (C terminal on insert)

Gene/Insert 2

  • Gene/Insert name
    BlastR
  • Tag / Fusion Protein
    • 2A (C terminal on insert)

Gene/Insert 3

  • Gene/Insert name
    EGFP

Resource Information

Depositor Comments

This lentiviral vector can be used as a standard for titering experiments when trying to compare vectors with different selection markers. It can also be used to determine if selection conditions with blasticidin or puromycin are using too high or too low a concentration of drug. If there are surviving cells that are EGFP-negative, then drug concentration is too low. If there are dying cells that express EGFP, then drug concentration is likely too high.

Information for Lentiviral Prep (Catalog # 59700-LV) ( Back to top )

Purpose

Ready-to-use Lentiviral Prep particles produced from pRosetta (#59700). In addition to the viral particles, you will also receive purified pRosetta plasmid DNA.

Lentiviral particles carrying a GFP tag and both puromycin and blasticidin resistance.

Delivery

  • Volume 1 mL
  • Titer ≥1x10⁶ TU/mL
  • Pricing $220 USD for preparation of 1 mL virus + $30 USD for plasmid.
  • Storage Store at -80℃. Thaw just before use and keep on ice.
  • Shipment Viral particles are shipped frozen on dry ice. Plasmid DNA (≥ 200ng) will also be included in the shipment.

Viral Production & Use

  • Packaging Plasmids psPAX2 (plasmid #12260)
  • Envelope pMD2.G (plasmid #12259)
  • Buffer DMEM +10% FBS
  • Selectable Marker Puromycin, Blasticidin

Biosafety

Requestor is responsible for compliance with their institution's biosafety regulations. Lentivirus is generally considered BSL-2. AAV is generally considered BSL-1, but may require BSL-2 handling depending on the insert. Biosafety Guide

Viral Quality Control

Titering Method:
  • Direct fluorescence: Lenti-X cells were transduced with serial dilutions of 59700-LV. 96 hours later GFP-positive cells were counted. You can view GFP expression in pRosetta-transduced cells here or at the image section at the top of this page. Read our fluorescence titering assay protocol here.
  • Colony formation: A549 cells were transduced with serial dilutions of 59700-LV and treated with blasticidin or puromycin. Blasticidin or puromycin-resistant colonies were expanded for approximately 2 weeks, stained with crystal violet, and counted.
Notes:
  • PCR confirmation of insert: PCR was carried out on the viral preparation with primers targeting the puromycin-resistance gene and the blasticidin-resistance gene. The PCR product was visualized on an agarose gel for size confirmation.
    Forward Primer: Puro-F2 CTCGGCTTCACCGTCACC
    Reverse Primer: Blast-R GCTCTTTCAATGAGGGTGGA

Visit our viral production page for more information.

How to cite this plasmid ( Back to top)

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.

  • For your Materials & Methods section:

    pRosetta was a gift from John Doench & David Root (Addgene plasmid # 59700)