Magnetic beads washing

DNA,83 which produces one million or more copies of amplified DNA in a short time. For identification of bacteria, PCR can be used to amplify DNA either after extraction from a sample or after lysis of the cells.83,84 Methods using washing, filtration, or magnetic beads with specific antibodies can be used to collect bacterial cells for PCR.85,86 PCR can be modified for the detection of bacteria from various sources32 and can even amplify DNA from dead cells.87... 

Figure 8 Chemiluminescent (A and B) and bioluminescent (C) detections for immobilized hybridizations of PCR product. Dg, digoxigenin Bt, biotin Ad, avidin. Procedure A [30] Biotin moiety is incorporated into PCR products during the amplification reaction, using one 5 -biotinylated primer. The product is hybridized with a Dg-labeled probe and is immobilized on streptavidin-coated magnetic beads. This capture reaction is carried out for 30 min at 37°C. A permanent magnet is used to sediment the beads during washing to remove unbound DNA. By incubation with the washed beads for 45 min at 37°C, anti-Dg antibody conjugated to HRP enzyme is bound to the Dg-labeled probe, and luminol reaction is performed for CL detection. Procedure B [31] Wells of apolystyrene microtiter plate are activated with l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and then coated with a labeled cDNA probe complementary to an internal region of the target DNA.

M sodium-phosphate solution pH 5 for washing and coating of the magnetic beads (solution A). 

After that, perform a washing step of the magnetic beads with 140 pL 5 x SSC for 10 min at 42°C. Repeat it again, eliminating the supernatant. 

Put the tubes on the magnetic separator until the solution becomes clear, and eliminate the supernatant. Perform a washing step of the magnetic beads with 150 pL of PBST. Repeat the washing step three times. 

ECL has also been applied to the analysis of polymerase chain reaction (PCR) products [53,55], PCR is first used to amplify the specific genes by use of two primers, one of which is biotinylated. The double-stranded DNA is then captured on streptavidin-coated magnetic beads and washed with an alkaline solution to denature and separate the strands. The particle-bound, single-strand DNA is used to capture products hybridized with Ru-labeled complementary... 

Magnetic beads (50 nm) were used in a PC chip to fractionate E. coli cells (K12) from sheep blood cells. A biotinylated rabbit polyclonal anti-E. coli antibody was first attached to the magnetic beads coated with streptavidin. Then E. coli became attached to the beads and was retained by the magnetic field. On the other hand, the other non-retained blood cells were washed away. The cell capture efficiency was evaluated after subsequent PCR of E. coli DNA [227]. 

Add 50 pL of washed magnetic beads suspension to 1 mL of cell suspension from step 2 and incubate at room temperature for 30 min on a rotator with end-over-end rotation, at 6 to 10 rpm. 

Remove the tube from the magnetic separation apparatus, carefully resuspend the cells in 10 mL PBS/10% FCS, and separate the cells coated with magnetic beads as in step 5. Repeat washing at least 3 times. 

Remove the storage buffer from 0.6 ml magnetic bead slurry (one tube) and wash 3 times with bead buffer (0.2 M NaCl, 20 mM Tris-HCl, 1 mM EDTA, pH 7.6). Resuspend beads in 625 ftl of this buffer and store at 4°. 

After the 6-min incubation, remove the NaOH solution (which contains the released, nonbiotinylated ssDNA strand) from the magnetic beads and add it to the ammonium acetate in the Centricon-100 tube. It is very important not to pipette any magnetic beads into the NaOH fraction. Most ssDNA will be recovered in this single NaOH wash. If desired, Steps 2 and 3 can be repeated. 

Ruthenium (II) tris(bipyridyl) (Figure 9-17, B) undergoes an electrochemiluminescent reaction (620 nm) with tripropylamine at an electrode surface, and this chelate is now used as a label in competitive and sandwich electrochemiluminescence immunoassays. Using this label, various assays have been developed in a flow cell using magnetic beads as the solid phase. Beads are captured at the electrode surface, and an unbound label is washed out of the cell by a wash buffer. Label bound to the bead undergoes an electrochemiluminescent reaction, and the light emission is measured by an adjacent photomultiplier tube. ... 

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