Positive displacement pipets

All reagents used for the PERT assay must be free of any contaminating MS 2-DNA. To avoid carryover contamination, set up at least two different rooms for work with materials before and after PCR. Use different sets of equipment, chemicals, and disposables. If possible, avoid the use of pH probes and spatula. If glass ware is used, do not have them washed in a central facility (where it can get contaminated), wash and bake it personally (at 240°C for 5 h). It may be a good idea to have a friend at a different location (where no work with MS2 is carried out) who can prepare solutions. Use only positive displacement pipetes or pipet tips with filters especially for PCR. The published recommendations in ref. 17 are useful. 

The second type of false-positive result can occur at the detection stage, e.g., when the liquid-transfer device pipets a strong positive sample followed by a negative sample. Because a typical PCR reaction can produce 10 -1012 molecules of amplified DNA in a 100-pl reaction and a radioactive probe can detect about 107 molecules, it is necessary to limit liquid carryover to less than 0.001-0.01 pi (Table 2). Such levels are possible with available automated pipeting devices, but manual pipeting should employ positive-displacement pipets or tips that prevent aerosols from contaminating the device. 

A third type of contamination is unique to PCR and other amplification methods, such as the ligase chain reaction. It involves the inadvertent contamination of a new reaction with the aerosolized products of a previous reaction. As shown in Table 2, as little as 10"7 pi of a tube of amplified DNA can contain 103 molecules of target (C4). Recommended precautions (K13) involve the use of positive-displacement pipets and the physical separation of areas where PCR reactions are analyzed from those where new reactions are setup. In laboratories that use these precautions, contamination is infrequent, and, when it does occur, is usually at the 1- to 100-molecule level. However, in addition to these procedural measures, it would be useful to have chemical or enzymatic methods of selectively inactivating amplified DNA—similar to the sterilization procedures used to inactivate large numbers of cultured viruses or bacteria. 

Detailed recommendations pertaining to equipment and supplies should also be given in the protocols. Positive-displacement pipets are crucial for successful PCR operations temperature-cycling instruments must meet certain minimum stan-... 

Figure 2.3 Schematic cross-sections of (a) air displacement pipet and (b) positive displacement pipet.

Quantify the retinoid stocks by UV spectroscopy. Use positive displacement pipets to accurately pipet ethanol (see Note 4) Discard the sample used in the spectrometer, as it may be photo-isomenzed... 

The setup and amplification section of a protocol also contains specific recommendations for the prevention of carryover of aerosolized DNA into the new reaction. Dedicated hoods or dead-air boxes are recommended in this step of the procedure. All pipets should be of the positive-displacement type. They should be kept in a dedicated setup hood and should never have previously been used to pipet amplified target. No amplified DNA should ever be brought into this area. During the reaction setup, either dUTP and UNG or isopsoralens may be added. 

Figure 17 (A) Schematic representation of bead deposition. (1) Drop of bead suspension is extruded out of a positive-displacement micropipet (2) after concentrating the beads at the bottom of the drop, the drop is reduced to 0.3 pL (3) the drop is set on the hydrophobic surface (4) the drop will stay at the pipet tip if the pipet is retracted slowly from the surface, leaving the beads behind. (B) SEM images of bead domains produced using 1 pL of (1) 4.1 X 10, (2) 4.1 X 1()6, (3) 4.1 X 10 beads/mL.

Figure 9.6 Manometer system for applying negative or positive pressures to the micropipet Coarse control is achieved by use of a simple syringe to displace volume above the front reservoir (on the right). Fine control, microatmospheres, is achieved by adjusting the position of the front reservoir using the sprung micrometer drive. The whole manometer system can be leveled with the pipet tip in order to achieve zero pressure at the pipet tip, using the central coarse screw. In-line pressure transducers measure small pressures (0-1000 dyne cm ) and large pressures (1000-100000 dyne cm ) [84].

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