InstaGeneTM Matrix - Bio-Rad Laboratories

1 InstaGeneTMMatrixCatalog # 03/17/2000 3:43 PM Page 2 Table of ContentsSection 1. Contents and 2. Instruction for DNA Preparation From Whole DNA Preparation From Cultured Mammalian DNA Preparation From Bacteria .. 03/17/2000 3:43 PM Page 31 Section 1. Contents and StorageThis bottle contains 20 ml of 6% instagene Matrix and a magneticstirbar. This is sufficient for 100 DNA preparations. Upon arrival, storethe Matrix at 4 WarningAvoid prolonged exposure of the Matrix to UV UseInstaGene Matrix allows fast and easy preparation of PCR*amplifiable DNA by eliminating labor intensive phenol/chloroformextraction steps.

2 A simple cell lysis step by boiling in the presence ofthe Matrix is sufficient. This is possible because the Matrix efficientlyabsorbs cell lysis products that interfere with the PCR amplificationprocess. Procedures for generating DNA suitable for PCR from wholeblood, cultured cells, and bacteria using instagene Matrix aredescribed on the following pages. Protocols for other types of tissuesor cells are presently being 03/17/2000 3:43 PM Page 42 Section 2. Instruction for DNA Preparation From Whole BloodThe protocol described below is for whole blood samples that arefrozen, refrigerated, or collected 3 6 l of whole blood to 1 ml of autoclaved nanopure water in ml microfuge tube.

3 Mix by inverting the tube several the tube at room temperature for 15 30 at 10,000 12,000 rpm for 2 3 remove all but 20 30 l of the supernatant. Do not disturbthe 200 l of instagene Matrix to the pellet and incubate at 56 Cfor 15 30 : instagene Matrix should be mixed at moderate speed on amagnetic stirrer to maintain the Matrix in suspension. The pipet tipused should have a large bore, such as a 1,000 l pipet tip( Bio-Rad s TBR-78 tip, catalog # 223-9378).6 Vortex at high speed for 10 seconds. Place the tube in a 100 C heatblock or boiling waterbath for 8 at high speed for 10 seconds.

4 Spin at 10,000 12,000 rpm for2 3 20 l of the resulting supernatant per 50 l PCR reaction. Storethe remainder of the supernatant at -20 C. Repeat step 7 whenreusing the instagene DNA :It is important to store the prepared sample at -20 :For more efficient DNA preparation from whole blood, theAquaPureTMGenomic DNA Blood Kit (catalog # 732-6345) 03/17/2000 3:43 PM Page DNA Preparation From Cultured Mammalian Cells1 Pellet 200 l of cell suspension from media in a microfuge tube. Spinat 10,000 12,000 rpm for 1 minute and remove the cells in 1 ml of 1x PBS and centrifuge for 1 minute at10,000 12,000 cells in autoclaved water at 20 30 cells/ 20 l of this cell suspension to 200 l of instagene at 56 C for 15 30 : instagene Matrix should be mixed at moderate speed on amagnetic stirrer to maintain the Matrix in suspension.

5 The pipet tipused should have a large bore, such as a 1,000 l pipet tip ( Bio-Rad s catalog # 223-9378).5 Vortex at high speed for 10 seconds. Place the tube in a 100 C heatblock or boiling waterbath for 8 at high speed for 10 seconds. Spin at 10,000 12,000 rpm for2 3 20 l of the resulting supernatant per 50 l PCR reaction. Storethe remainder of the supernatant at -20 C. Repeat step 6 whenreusing the instagene DNA :It is important to store the prepared sample at -20 03/17/2000 3:43 PM Page DNA Preparation From BacteriaThe protocol described below is for the preparation of genomic DNAor episomal DNA from an isolated bacterial colony and resuspend it in 1 ml ofautoclaved water in a microfuge for 1 minute at 10,000 12,000 rpm.

6 Remove 200 l of instagene Matrix to the pellet and incubate at 56 Cfor 15 30 : instagene Matrix should be mixed at moderate speed on amagnetic stirrer to maintain the Matrix in suspension. The pipet tipused should have a large bore, such as a 1,000 l pipet tip ( Bio-Rad s catalog # 223-9378).4 Vortex at high speed for 10 seconds. Place the tube in a 100 C heatblock or boiling waterbath for 8 at high speed for 10 seconds. Spin at 10,000 12,000 rpm for2 3 20 l of the resulting supernatant per 50 l PCR reaction. Storethe remainder of the supernatant at -20 C.

7 Repeat step 5 whenreusing the instagene DNA :It is important to store the prepared sample at -20 C.*The polymerase chain reaction (PCR) process is covered by patent numbers4,683,195, 4,683,202, and 4,899,818 which are owned by Hoffman-La Roche, Inc. andF. Hoffman- La Roche, Ltd. The purchase of this product does not convey a license touse the process covered by these patents. The user of this product to perform PCRmust obtain a license from Hoffman-La Roche, 03/17/2000 3:43 PM Page 7 Bio-Rad Laboratories2000 Alfred Nobel Dr., Hercules, CA 94547510-741-1000 LIT544 Rev 03/17/2000 3:43 PM Page 1