"In-gel" purified ditags direct synthesis of highly efficient SAGE Libraries

BACKGROUND: SAGE (serial analysis of gene expression) is a recently developed technique for systematic analysis of eukaryotic transcriptomes. The most critical step in the SAGE method is large scale amplification of ditags which are then are concatemerized for the construction of representative SAGE libraries. Here, we report a protocol for purifying these ditags via an 'in situ' PAGE purification method. This generates ditags free of linker contaminations, making library construction simpler and more efficient. RESULTS: Ditags used to generate SAGE libraries were demarcated 'in situ' on preparative polyacrylamide gels using XC and BPB dyes, which precisely straddle the ditag band when a 16% PAGE gel (19:1 acrylamide:bis, 5% cross linker) is used to resolve the DNA bands. Here, the ditag DNA was directly excised from gel without visualization via EtBr or fluorescent dye staining, resulting in highly purified ditag DNA free of contaminating linkers. These ditags could be rapidly self ligated even at 4°C to generate concatemers in a controlled manner, which in turn enabled us to generate highly efficient SAGE libraries. This reduced the labor and time necessary, as well as the cost. CONCLUSIONS: This approach greatly simplified the ditag purification procedure for constructing SAGE libraries. Since the traditional post-run staining with EtBr or fluorescent dyes routinely results in cross contamination of a DNA band of interest by other DNA in the gel, the dry gel DNA excision method described here may also be amenable to other molecular biology techniques in which DNA purity is critically important

A Lab Assembled Microcontroller-Based Sensor Module for Continuous Oxygen Measurement in Portable Hypoxia Chambers.

Hypoxia-based cell culture experiments are routine and essential components of in vitro cancer research. Most laboratories use low-cost portable modular chambers to achieve hypoxic conditions for cell cultures, where the sealed chambers are purged with a gas mixture of preset O2 concentration. Studies are conducted under the assumption that hypoxia remains unaltered throughout the 48 to 72 hour duration of such experiments. Since these chambers lack any sensor or detection system to monitor gas-phase O2, the cell-based data tend to be non-uniform due to the ad hoc nature of the experimental setup.With the availability of low-cost open-source microcontroller-based electronic project kits, it is now possible for researchers to program these with easy-to-use software, link them to sensors, and place them in basic scientific apparatus to monitor and record experimental parameters. We report here the design and construction of a small-footprint kit for continuous measurement and recording of O2 concentration in modular hypoxia chambers. The low-cost assembly (US$135) consists of an Arduino-based microcontroller, data-logging freeware, and a factory pre-calibrated miniature O2 sensor. A small, intuitive software program was written by the authors to control the data input and output. The basic nature of the kit will enable any student in biology with minimal experience in hobby-electronics to assemble the system and edit the program parameters to suit individual experimental conditions.We show the kit's utility and stability of data output via a series of hypoxia experiments. The studies also demonstrated the critical need to monitor and adjust gas-phase O2 concentration during hypoxia-based experiments to prevent experimental errors or failure due to partial loss of hypoxia. Thus, incorporating the sensor-microcontroller module to a portable hypoxia chamber provides a researcher a capability that was previously available only to labs with access to sophisticated (and expensive) cell culture incubators

Oxygen and temperature changes during programmed adjustment of hypoxia.

<p>A single 24-well plate of cell cultures was used and monitored over 48 hrs. (<b>A</b>) The sealed modular hypoxia chamber was placed in cell culture incubator (without a purge with anoxic gas-mix). The chamber was then flushed with the anoxic gas-mix at an initial flow rate of 20 L/min (approx. 2 min) to lower the O₂ level from 21% to 5%. This was followed by a slower flow rate of 5 L/min (approx. 3 min) until O₂% reached 0.5%. (<b>B</b>) expanded view of the change in O₂% during the initial 5 min programmed purge procedure.</p

Required electronic parts^a and accessories^b.

<p>Required electronic parts<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148923#t001fn001" target="_blank">^a</a> and accessories<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148923#t001fn002" target="_blank">^b</a>.</p

Screen-capture images of CoolTerm terminal window.

<p><b>Upper panel</b>: CoolTerm quick access bar and ribbon; <b>middle panel</b>: format of the initial data output from CoolTerm; <b>lower panel</b>: format of the final data saved by CoolTerm with time-stamps (saved and opened as a Microsoft Notepad file).</p

Representative oxygen and temperature data transmitted by the sensor under experimental conditions.

<p>(<b>A</b>) change in chamber O₂% upon a single 4 min (20 L/min) anoxic gas purge conducted in the presence of cell cultures. (<b>B</b>) lack of a rapid initial increase in O₂% when chamber is devoid of any cell cultures, liquid media or water. (<b>C</b>) change in chamber O₂% with a post-1 day gas purge (in the presence of cell cultures). (<b>D</b>) change in chamber O₂% with daily gas purges in the presence of cell cultures. (<b>E</b>) pressure profile of experiment listed in <b>D</b>. (<b>F</b>) change in chamber O₂% profile upon inclusion of ten cell culture plates.</p

Schematic of the logic converter and connections.

<p>Pin designations of the 4-channel bi-directional logic converter (low-voltage pins A1, A2 and high voltage pins B1, B2 were used. A3, A4 and B3, B4 pins are extra, and not needed in this application). The logic converter is supplied as a mini printed circuit board (PCB) with a pair of 6-pin headers to facilitate mounting on a breadboard. We used a 30W soldering iron (RadioShack) fitted with a 15-watt soldering iron tip (cat. no. 64–2052, RadioShack) and 0.787 mm (0.032") flux-cored solder wire (cat. no. 64–005) to solder the PCB to the 6-pin headers. The soldered PCB was fitted on the half-sized breadboard mounted alongside the Arduino board.</p

Oxygen sensor-hypoxia chamber module.

<p>(<b>A</b>) Close-up views of the Arduino Uno microcontroller board and half-sized breadboard mounted on acrylic plate. <b>(B)</b> Layout of the modular hypoxia chamber (with the oxygen sensor mounted on half-sized breadboard), the Arduino Uno microcontroller and the wire harness routed through one of the gas-flush tube ports, before assembly of the module (lid not shown). 30 AWG silicone-coated (orange colored) wires (cat. no. 2001, adafuit.com) were used in this photograph to clearly illustrate the wire harness.</p

Outline of the wiring diagram.

<p>A Fritzing (fritzing.org) sketch is presented outlining the wiring connections between the microcontroller, the logic converter, and the oxygen sensor.</p