TIP2k-Module

From Bioblast
Jump to: navigation, search

Oroboros Instruments

News         O2k-Products         O2k-Purchase         O2k-Open Support         O2k-Publications         O2k-Workshops and Events         O2k-Network         O2k-Feedback         NextGen-O2k



TIP2k-Module

O2k-Catalogue

Description TIP2k-Module - Titration-Injection microPump (TIP2k) for two-channel operation with the O2k-FluoRespirometer with automatic control by DatLab of programmable titration regimes and feedback control (oxystat, pH-stat). The TIP2k-Module includes TIP2k-Accessories: 4 Microinjection syringes, 1 Pkg. TIP2k-Needle Spacers with mounting tool, 1 TIP2k-Needle Safety Support and 1 Pkg. TIP2k Filter Papers, and the newest DatLab version.
Product ID 11100-03
Type O2k, O2k-Module, Microsyringe, O2k-FluoRespirometer
Link MiPNet12.10 TIP2k-manual, MiPNet04.05 Titration-Injection
Image
TIP2k with 200 mm3 microsyringe.JPG


MitoPedia O2k and high-resolution respirometry: O2k hardware 

TIP2k-Module consists of:

TitleDescriptionProduct idProduct image
Titration-Injection microPumpTitration-Injection microPump (TIP2k) for two-channel operation with the O2k with automatic control by DatLab of programmable titration regimes and feedback control (oxystat, pH-stat). Included in the TIP2k-Module.31000-01
Titration-Injection-microPump.jpg
Microsyringe\200 mm3\TIP2kMicrosyringe\200 mm3\TIP2k: Microinjection syringe for Titration-Injection microPump, 200 mm3 (µl), fixed injection needle with rounded tip, with spacers. Four units of this item are standard components of the TIP2k-Module.31220-01
Microsyringe200 mm3TIP2k.JPG
TIP2k-Needle SpacerTIP2k-Needle Spacers for microinjection TIP2k needle, silicone stops (200/Pkg.) with mounting tool. This item is a standard component of the TIP2k-Module.31310-01
80600-24 Spacers f. TIP2k needle.JPG
TIP2k Filter PapersTIP2k Filter Papers (package of 10) to be used for the stoppers during TIP2k titration for Instrumental oxygen background correction to avoid constant manual siphoning off of extruded liquid. One package is a standard component of the TIP2k-Module.31320-01
TIP2k Filter Papers.JPG
TIP2k-Needle Safety Support with cable guideTIP2k-Needle Safety Support with cable guide: for safe storage of TIP2k-needles and MultiSensor Modules calbes when not required during the experiment. This item is a standard component of the TIP2k-Module, O2k-TPP+ ISE-Module, O2k-pH ISE-Module.31330-02
31330-01 3.jpg
Template NextGen-O2k.jpg
O2k-Open Support


MitoPedia O2k and high-resolution respirometry: O2k-Open Support 

O2k-Open Support

The TIP2k provides unique advantages for applications which require highest accuracy of titration volumes and automatic control of complex titration-injection protocols. The TIP2k was specially developed for the O2k. The main modes of operation are automatic multi-step injections, continuous injections for steady-state respirometry, (both operated form the direct control mode), and feedback controlled operation.
Direct Control Mode: Operation mode for delivering a defined volume in a short pulse. A number of cycles, N, can be defined, and the duration of the interval from injection to injection determined, repeating the process N times. Several such steps can be programmed to run in sequence to give an entire TIP2k program.
Feedback Control Mode: Operation mode to reach and maintain a certain parameter (e.g. oxygen concentration) by automatic TIP injections.
» TIP2k-Manual - O2k-Manual for using the Titration-Injection microPump and programming TIP2k setups in DatLab.
The TIP2k is fully supported by the O2k.
The TIP2k is placed at the top of the Oroboros O2k. It does not need any extra bench space.
  • Align the front legs of the TIP2k with the TIP2k-Spacer Bar on the front of the O2k if you use 200 µl syringes.
  • Align the rear ends of the TIP2k and O2k, if you use 500 µl syringes.

Technical characteristics

Titration: Programmable, automatic titration regimes, with titration volumes of 0.05 to 250 µl, variable titration intervals and duration of titration pulses.
Injection: Steady-state injection: Operation at quasi steady-states by continuous injection of substrates at limiting rates of consumption, providing new flexibility in experimental design by combining the technical advantages of closed and open systems. Programmable injection flows: 0.01 to 25 µl.s-1.

Ramp injection (MiPNet04.05, see above): Ramp increase of effector concentrations by "continuous titration".

  • DatLab software for feedback control by the the TIP2k: for steady-state respirometry at selected oxygen levels and pH-stat applications.

TIP2k-Syringes

» SOP: Handling microsyringes
  • Rinse the outside with water immediately after use.
  • Wash 3 x with last used solvent, rinse with EtOH.
  • Storage: ethanol (short), dry (longtime).
  • Rinse with pure solvent before use.

Cleaning TIP2k-syringes

  1. To prevent washing out effects when the needle is inserted into the chamber the inner diameter of the TIP needle has to be very small. This means that the needle is easily blocked by small particles either precipitated from the solution or introduced with the solution.
  2. While a TIP syringe has to be considered a consumable, it is possible to lengthen its lifetime by carefully following the procedures suggested in the TIP-2k Manual.
    1. The main way to prevent clogging of needles is to follow rigorously the cleaning procedure after each usage.
    2. Additionally, it has to be taken care that no particles are suspended in the used solutions.
    3. For not very well soluble substances, filtering of the solution may be necessary. Small particles can also be introduced by laboratory activities, e.g. from paper towels or from powdered gloves. Only use powder-free gloves when working with TIP syringes!
  • Examples for proper TIP use and cleaning protocols
  1. Instrumental O2 background (dithionite in phosphate buffer or MiR05)
    1. Clean (fill and empty) the syringe with water.
    2. Fill the syringe with dithionite solution for washing, discard immediately.
    3. Fill the syringe with dithionite solution (in buffer) for use during the assay.
    4. After the assay empty the syringe and immediately wash the outside of the needle with water (because there is medium sticking to the outside).
    5. Wash (fill and empty cycles) the syringe three times with water.
    6. Store the syringe (will be partially filled with water) or after cleaning with ethanol (then it will be partially filled with ethanol.
  2. CCCP titration (CCCP dissolved in ethanol)
    1. Clean (fill and empty cycle) the syringe with pure ethanol.
    2. Clean with CCCP solution (in this case more important than in example 1 because you want to have a precise CCCP concentration).
    3. Fill with CCCP stock solution and start the experiment.
    4. After the assay empty the syringe and immediately wash the outside of the needle with water (because there is medium sticking to the outside)
    5. Clean (fill and empty cycles) the syringe three times with ethanol.
    6. Store the syringe (will be partially filled with ethanol) or wash it with water and then store (then it will be partially filled with water).


Programming the TIP2k in DatLab

Programming the TIP2k for "BG_Feedback"

In the TIP2k window the modus can be changed by clicking on either direct control or feedback control. In the feedback control several lines can be inserted, all of which take effect during the currently selected line of the main program. In the figure the implementation of program line 1 of the example set up is shown.
In the main window (Program line), blue highlighting shows that the first program line is selected. The first line in the feedback control window (upper right corner) sets the "start injection" value to 120 mM. The injection is only started when this condition is valid for two consecutive data points (DataN = 2). The interval is set to 200 seconds, which equals the maximum injection time when the maximum volume is set to 100 µl. These parameters are not important for the discussed purpose; however the interval has to be set to at least the (maximum) injection time calculated at the left side. Once an injection is started the program checks the following lines for "stop" instructions. If just one of these conditions is met, the injection will stop. In our example a stop condition is set at 100 µM.


TIP2k Volume + Time

Many thanks to Therkelsen AIb from DK Copenhagen Quistorff B for pointing out this problem.
  • Problem: If the user programs an injection by setting volume and time (button "Vol+Time") the required flow is calculated by DatLab. The result is rounded to a precision that can be handled by the TIP2k. For long, slow injections this may result in significantly more or less time being necessary to inject the desired volume than the time entered by the user. If this injection is immediately or with a short delay followed by another injection this may lead to a conflict between the not finished first and the already starting second injection. Using DatLab 4 in this situation will crash the TIP program (error "TIP already running"). In DatLab 6 the program will be executed with the start of the second injection delayed. However the TIP2k status line will behave strangely in the overlapping time and events may be set at the wrong time.
  1. Suggestions: Use the rounded flow as a starting point to set you own Flow + Volume or Flow + Time values that fit your experimental design.
  2. Alternative, not tested for all DatLab versions: After entering the desired volume and time, press either the button "Vol+Flow" or "Flow+Time". DatLab will use the rounded flow to calculate a new time, or a new volume, respectively.

TIP2k errors

  • O2k does not recognize the TIP2k
  • TIP2k is not responding
  • No lights on the TIP2k
Solutions
  1. Check if your O2k has the upgrade for your TIP2k
  2. For O2k Series E onwards check if you have connected the TIP2k cable to the plug-in TIP1

List of publications: TIP2k