Pipets multichannel

The siRNA libraries are obtained as a 10 pM stock and we prepare a 1 30 dilution resulting in 333 nM daughter plate concentrations. Although this step can be done manually using multichannel pipets, it can be greatly expedited by the use of a liquid-handling system, such as the Biomek EX Workstation (Beckman Coulter Brea, CA). 

Add 20 pL 10% MPBS and 80 pL supernatant to each well Then immediately, using a multichannel pipet, add 12 pL freshly prepared developing reagent (e g., for myc-tag, containing antibodies 9E10 [50 pg/mL] and antimouse HRP [ 1 100] in 2% MPBS)... 

Instrument precision is a measure of reproducibility, i.e., how consistent an instrument is in delivering volume. This is generally measured as a coefficient of variance (CV). Reproducibility for multichannel (96- or 384-tip) pipetting instruments has two aspects. Within-plate variability measures the consistency from one channel to another in the same transfer cross-plate variation measures the consistency of the same channel through multiple transfers. A good liquid handler should be consistent in both functions. 

Prepare a cell suspension in fresh growth medium from a flask of cells in exponential growth and count cells. Dilute cells to density of 104 cells /mL (see Note 1) allowing 30 mL of cell suspension for 3 plates. Transfer the cell suspension to a 10 cm Petri dish and with a multichannel pipet add 100 pL to the central 10 columns of a round bottomed microtitre plate. Add 200 pL of growth medium to the wells in columns 1 and 12. Put plates in a plastic box and incubate in a humidified atmosphere at 37°C while drug solutions are prepared. 

DMSO can damage some multichannel pipets. Costar pipettes are essentially resistant, but DMSO should still be dispensed with caution. 

The plate is placed in the plate reader and the enzyme recycling reaction initiated by the addition of glutathione reductase, 40 pL to each well, again using a multichannel pipet. Care should be taken to ensure that there are no bubbles in the wells. 

Place the plates on a rocking platform for 20 min at room temperature. Complete cell lysis by pipetting the contents of each well several times with a multichannel pipeter. 

Mix samples by pipeting the contents of each well several times with a multichannel pipeter. 

Stop the reaction by adding 150 pL 1M calcium carbonate to each well. Mix the contents of the wells with a multichannel pipeter. 

The volumes of the helicase reaction in the assay were dictated in part by the requirements of multichannel pipets and robotic equipment for quick and reproducible handling. Calculate the total amount of helicase reaction mix needed for each run of the assay and prepare one large batch. Factor in extra reactions to account for loss of the mix on pipet tips, tubes, and so on. 

Add 5 pL of the culture supernatants into the individuals wells containing the radioactive RT assay solution. Duplicate reaction wells for each supernatant are recommended. If the culture supernatants are stored in a 96-well plate, a small-volume multichannel pipeter can be conveniently used for this addition. 

Using a multichannel pipeter, the coated plate is gently washed (twice) with sterile PBS. 

Using a multichannel pipeter the growth medium is gently removed from the cells. 

Using a multichannel pipet, carefully remove 150 pL growth medium. 

On d 4, microscopically evaluate the PBMCs. Resuspend the cells in the wells and remove 125 pL from each well, using a multichannel pipet. Move from the lowest concentration to the highest concentration. 

On d 7, aliquots of 100 pL supernatant are carefully taken using a multichannel pipet. These samples are stored at -80°C until p24 antigen ELISA is performed. 

Make nine serial, fivefold dilutions of the virus directly in the microtiter trays using the Biomek 2000 robot or a multichannel pipet take 25 pL out of column 2 and add it to column 3, mix, change tips, and again take 25 pL out of column 3 and repeat the dilution up to column 10. Leave row 11 without vims, as control (see Table 4). 

Resuspend the MT-4 cells at 3 x 105 cells/mL complete medium in a flask that is connected with an autoclavable dispensing cassette of a Titertek Multidrop dispenser (or use a multichannel pipet). 

Add to each well (except the outer row) of the microtiter trays 20 pL of MTT (7.5 mg/mL) using the Titertek Multidrop (or a multichannel pipet). 

Remove a constant volume of medium (e.g., 150 pL) from each cup using the Biomek 2000 robot (or a multichannel pipet) without disturbing the MT-4 cell clusters containing the formazan crystals. 

As an alternative, heat-resistant microtiter plates and corresponding thermal cycling machines can be used. This increases the capacity of reactions that can be analyzed and allows easy transfer of samples with a multichannel pipet after amplification. 

Reservoirs for multichannel pipetting of buffers and so forth (Costar, Cambridge, MA). 

As manual, multichannel pipeting devices and plate-based detection systems became staple tools in the modem discovery laboratory, bioassays for moderate throughput have been developed in a wide variety of formats. The flexibility of these detection devices allowed assays that utilize visible-, fluorescence-, luminescence-, and radioactive-based readouts. Manual pi-... 

Figure 3 Robot layout. A typical operating scheme may be as follows. The robotic arm picks up a plate from the sample station, brings it to the barcode reader and puts it on the working area. The robotic arm then grabs the 96-well multichannel pipettor from the equipment station, pipets reagents from the reagent station, and dispenses them into the microtiter plate in the working area. The plate is agitated and placed in the incubator. The same sequel of steps is then performed on another plate. After the required incubation time, the robotic arm transports the microtiter plate to the barcode reader and then to the detector.

Usually a microtiter plate well. Specially prepared ELISA plates are commercially available. These have an 8 jA 12 well format and can be used with a wide variety of specialized equipment designed for rapid manipulation of samples including multichannel pipets. 

The multichannel pipets used in the ELISA can be used to fill the wells carefully. The washing solution is contained in reservoirs. ... 

The microtiter plate system is ideally used in conjunction with multichannel microtiter pipets. Essentially they allow the delivery of reagents via 4, 8, or 12 channels and are of fixed or variable volumes of 25"C250 pL. 

Accuracy and precision can easily be calculated by the formulae in Subheadings 3.2.2.I. and 3.2.2.2. In contrast to commercial pipet calibration computer software, the conversion factor for calculating the density of water suspended in air at the test temperature and pressure are not considered. For calibration of multichannel pipets, examine each channel separately. 

---