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SapphireTM Insect Transfection Reagent

nsect cells and lytic baculoviruses provide a proven method for highlevel expression of full-length mammalian proteins. Autographa californica nuclear polyhedrosis virus (AcNPV) is used to infect cultured insect cells (e.g. Spodoptera frugiperda). Expression of the highly abundant polyhedrin gene is nonessential in tissue culture and its strong promoter can be used for the transcription of foreign genes. The polyhedrin promoter is maximally expressed very late stage in infection when the lytic virus kills the host cells, resulting in high levels of expression even for certain toxic proteins. In addition, many post-translational modifications similar to those in mammalian cells are made in insect cells and proteins unable to be expressed in E. coli have been successfully expressed in the insect cell system. The Sapphire Insect Transfection Reagent is for easy and efficient transfection of DNAs into insect cells. It is optimized for use with Sapphire Baculovirus DNA and various transfer vectors.

content of tube containing transfection mix slowly into one containing DNA. 4. Let mixture sit for 10-15 min at on ice and bring the volume to 1 ml total by adding TNMFH minus serum. 5. Remove media from 35 mm dish and add the combined mixture from Step 4 dropwise to cells from Step 2. For negative control, use Sapphire DNA alone without any vector DNA. For positive control, use pVL1392XylE (Cat. No. ABP-BVP-10001) in combination with Sapphire DNA. 6. Incubate the plates at 27C for 4-5 hours. After that time, remove medium and replace it with 2 ml of fresh TNM-FH medium containing 10% fetal bovine serum (Cat. No. ABP-MED-10001). 7. After 5 days, collect the supernatant of all three plates and infect fresh cells to amplify the virus. Optionally, lyse the transfected cells and check for expression of your protein of interest.

or Research Use Only. Not for Diagnostic or Therapeutic Use. Purchase does not include or carry any right to resell or transfer this product either as a stand-alone product or as a component of another product. Any use of this product other than the permitted use without the express written authorization of Allele Biotech is strictly prohibited

Website: www.allelebiotech.com Call: 1-800-991-RNAi/858-587-6645 (Pacific Time: 9:00AM~5:00PM) Email: oligo@allelebiotech.com

Protocol
Transfection Using the Sapphire Transfection Reagent
1. Seed 1 x 106 Sf9 (Cat. No. ABPCEL-10001) or Sf21 (Cat. No. ABPCEL-10002) cells onto each 35mm tissue culture plate. Allow cells to attach firmly which takes usually 5 to 10 min. Note: Prepare three plates for each co-transfection: one for the positive control, one for the negative control, and one for the recombinant plasmid of interest. 2. Remove medium and replace it with 1 ml of sterile TNMFH minus serum. 3. Mix 0.1 g of Sapphire baculoviral DNA and 1g of recombinant baculovirus transfer vector containing gene of interest in a sterile microfuge tube containing 100 ul of TNMFH minus serum. In a separate tube prepare transfection mix containing 3 l of Insect shuttle transfection reagent and 97 l of TNMFH minus serum. Combine both tubes by pipetting the

Box 1 | Product Summary


Catalogue Number Component Storage Stability ABP-BVD-10003 75 l of DNA insect shuttle transfection reagents (25 reactions) Store at +4C. The transfection buffers are stable for six months when stored properly.

Box 2 | Related Products


Product
Sapphiretm Baculovirus DNA (10rxn) Sapphire Baculovirus DNA and Transfection Reagent Sf9 Cells (frozen, 107 cells) Sf21 Cells (frozen, 107 cells) T.ni Cells (frozen, 10 cells)
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Catalogue No.
ABP-BVD-10001 ABP-BVD-10002 ABP-CEL-10006 ABP-CEL-10007 ABP-CEL-10008 ABP-MED-10001 ABP-MED-10002 ABP-MED-10004 ABP-BVP-10001 ABP-BUF-10010

TNM-FH Insect Culture Medium Serum Free Insect Culture Medium Graces Insect Culture Medium pVL-1392-XyIE Control Vector Insect Lysis Buffer 5X (10ml)

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