Prepare DNA from blood, cultured cells, or bacteria:
Just incubate sample with InstaGene matrix at 56C for 1530 min, then boil for 8 min.
InstaGene matrix absorbs cell lysis products, producing an improved substrate for PCR amplification.
DNA Preparation From Whole Blood
Use whole blood samples that are frozen, refrigerated, or collected fresh.
1. Add 36 l of whole blood to 1 ml of autoclaved double distilled water in a 1.5 ml microfuge tube. Mix by inverting the tube several times.
2. Incubate the tube at room temperature for 1530 min.
3. Spin at 10,00012,000 rpm for 23 min.
4. Carefully remove all but 2030 l of the supernatant. Do not disturb the pellet.
5. Add 200 l of InstaGene matrix to the pellet and incubate at 56C for 1530 min.
Note: InstaGene matrix should be mixed at moderate speed on a magnetic stirrer to maintain the matrix in suspension. The pipet tip used should have a large bore, such as on a 1,000 l pipet tip (Bio-Rads TBR tips, catalog #223-9350 and 223-9351).
6. Vortex at high speed for 10 sec. Place the tube in a 100C heating block or boiling water bath for 8 min.
7. Vortex at high speed for 10 sec. Spin at 10,00012,000 rpm for 23 min.
8. Use 20 l of the resulting supernatant per 50 l PCR reaction. Store the remainder at -20C. Repeat step 7 when reusing the InstaGene DNA preparation.
Note: It is important to store the prepared sample at -20C when not in use.
DNA Preparation From Cultured Mammalian Cells
1. Pellet 200 l of cells suspension from media in a microfuge tube. Spin at 10,00012,000 rpm for 1 min and remove the supernatant.
2. Resuspend cells in 1 ml of 1x PBS and centrifuge for 1 min at 10,00012,000 rpm.
3. Resuspend cells in autoclaved water at 2030 cells/l.
4. Add 20 l of this cell suspension to 200 l of InstaGene matrix. Incubate at 56C for 1530 min.
Note: InstaGene matrix should be mixed at moderate speed on a magnetic stirrer to maintain the matrix in suspension. The pipet tip used should have a large bore, such as on a 1,000 l pipet tip.
5. Follow the procedure for DNA preparation from whole blood, steps 68.
DNA Preparation From Bacteria
The protocol described below is for the preparation of genomic DNA or episomal DNA from bacteria.
1. Pick an isolated bacterial colony and resuspend it in 1 ml of autoclaved water in a microfuge tube.
2. Centrifuge for 1 min at 10,00012,000 rpm. Remove the supernatant.
3. Follow the DNA preparation procedure for whole blood, steps 58.
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