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pSLIC-PuroP2A™ Lentivirus Plasmid Construction Kit ( LT2002)

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USD$400.00
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Quick Overview

Download: USER MANUAL PDF | MSDS PDF


ATCGbio Life Technology Inc. provides pSLIC-PuroP2ATM lentivirus plasmid construction kit (LT2002) to let user creates the lentivirus plasmid for the gene of interest. This kit comes with SLIC-EnzymeTM(SE1001-01) and a lentivirus plasmid which contains puromycin resistance gene and P2A sequence.


SLIC (sequence and ligation independent cloning) allows users to insert PCR product into the plasmid without ligation. P2A peptide sequence allows to express both puromycin resistance gene and user’s gene of interest by a single CMV promoter (bicistronic). P2A sequence in our plasmid is GSGATNFSLLKQAGDVEENPG/PD. The first part (before slash "/ ") GSGATNFSLLKQAGDVEENPG is attached to puromycin resistance gene product and the second part (after slash "/ ") PD is attached to the gene of interest users insert. 


Our proprietary SLIC-EnzymeTM system which has superb performance and temperature stability (stable even at room temperature for a few days). In 30min reaction with SLIC-EnzymeTM, PCR product is inserted downstream of P2A sequence.

pSLIC-PuroP2A™ Lentivirus Plasmid Construction Kit ( LT2002)

Details

 Kit components: shipped at room temperature, and stored at following temperature.

Catalog 
 
Components  
Size 
Storage
LT2002-01
 
pSLIC-PuroP2ATM Plasmid
20µl (200ng/µl)
4°C.This plasmid is not intended to amplify.
SE1001-01
 
10×SLIC-EnzymeTM
25µl
-20°C, Enzyme is stable at least 4 days at room temperature.
GFP1001-01
 
GFP Template DNA (control)
10µl (10ng/µl)
4°C
GFP1002-01
 
GFP Forward and Reverse
Primer Mixture
10µl, 10µM 
4°C 

The kit contains enough plasmids and reagents to perform at least 20 reactions for creating lentiviral vector plasmids which contain gene of interest.

pSLIC-PuroP2ATM structure

pSLIC-PuroP2Astructure2

ATCGbio Life Technology Inc. provides pSLIC-PuroP2ATM lentivirus construction kit (LT2002) to let user creates the lentivirus plasmid for the gene of interest. This Kit comes with SLIC-EnzymeTM and a lentivirus plasmid which contain puromycin resistance gene and P2A sequence. SLIC-EnzymeTM allows users to insert PCR product into the plasmid without ligation. This technique has been developed last 10 years (for example, see Nature Methods 4:251-256, 2007) and we created our proprietary SLIC-EnzymeTM system which has superb performance and temperature stability (stable even at room temperature for a few days). P2A peptide sequence allows to express both puromycin resistance gene and user’s gene of interest by a single CMV promoter (bicistronic).  To make the ligation effective, a staffer sequence was inserted between EcoR V., user first digests the plasmid with EcoR V and then inserts the PCR product by SLIC-EnzymeTM reaction.  User performs PCR (gene of insert) with primers having 15bp homologous to the ends of EcoR V sequence. In 30 min reaction with SLIC-EnzymeTM, PCR product is inserted downstream of P2A sequence.

P2A-like peptide sequence was discovered in various virus genomes and found to self-cleave near the end of sequence during translation.  P2A sequence in our plasmid is GSGATNFSLLKQAGDVEENPG/PD. The first part (before slash ”/”) GSGATNFSLLKQAGDVEENPG is attached to puromycin resistance gene product and the second part (after slash "/") PD is attached to the gene of interest users insert. Start codon of gene of interest (ATG) may be removed. Alternatively, user can put flag-tag (DYKDDDDK) sequence at the N-terminus gene of interest just by a single PCR as shown in this manual.

pSLIC-PuroP2Amap

Our pSLICs plasmids backbone structure size is 6.8Kb plasmid having 3.4 kB basic lentivirus components between 2 LTRs. Original 5’ LTR promoter activity will be self-inactivated during virus production. pSLIC-PuroP2ATM has the puromycin resistance gene with P2A sequence (total ~0.7 kB) followed by a staffer sequence flanked by EcoRV site.

Gene of interest should be subcloned by PCR by primers containing 15bp homologous to end of EcoR V digestion site (see above figure). Red colored sequences need to be incorporated into PCR product. The PCR product should be gel-purified. By SLIC-EnzymeTM reaction, PCR fragments can be inserted to pSLIC-PuroP2ATM plasmid in 30 minute.

For more details, please read the User Manual.

In Vitro Expamle

The HepG2 cells were infected with lentivirus expressing GFP made by pSLIC-PuroP2ATM kit using our Lentivirus Production KitTM, LT1001 (the amount of lentivirus used was produced in only one well of 6-well plate).

    Light image (bar=200μm;×100 amplification)           Fluorescence image (bar=200μm; ×100 amplification)
pSLIC-PuroP2A infect HepG2 cells 1     pSLIC-PuroP2A infect HepG2 cells 2
Expression of GFP were observed in 24 hrs after infection. At 72hrs post-infection, selection was done with 0.5µg/ml puromycin overnight incubation followed by replate the cells containing 0.5 µg/ml puromycin medium. The pictures were taken 2 days after re-plate. Almost all the cells were positive for GFP.

References

Y. Ido, et al. PLoS ONE, 2012 Apr 07(4): e35092

Mamie Z Li & Stephen J Elledge  Nature Methods 2007; 4:251-256

Gibson DG Methods Enzymol 2011; 498:349-61

Jin Hee Kim, Sang-Rok Lee, Li-Hua Li, et al.  PLoS ONE 2011 Apr 06 (4): e18556

Georgios Trichas, Jo Begbie and Shankar Srinivas  BMC Biology 2008, 6:40

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