HybriCycler Hybridization Oven Operating Instructions IMPORTANT: Please read these instructions before operating your UVP HybriCyclerTM system to familiarize yourself with operation procedures.
HC-3000 HybriCycler Hybridization Oven 2 Introduction The HybriCycler Hybridization Oven is designed as a compact alternative for hybridization requirements. The HybriCycler Oven features microprocessor operation for precise mixing capability. This space-saving design is manufactured with a 3/8” thick beta-blocking acrylic and polycarbonate cylinder providing 360° view of the bottles.
HC-3000 HybriCycler Hybridization Oven HybriCycler Hybridization Oven Hardware The HybriCycler Oven is comprised of: Hybridization Chamber The space-saving Hybridization Chamber is manufactured of 3/8” thick beta-blocking acrylic providing a 360° view of the bottles. Bottle Carousel The cartridge-style bottle carousel is manufactured of ¼” thick polycarbonate.
HC-3000 HybriCycler Hybridization Oven 4 Set-Up Instructions This equipment is not intended for interconnection with any other devices. Use of this equipment other than intended may create a safety hazard and/or malfunction. 1. Plug the female end of the power cord to the connection on the back of the HybriCycler. 2. Insert your Hybridization bottle(s) into the bottle carousel. Start by sliding the bottle into the bottle carousel’s entry hole at either end of the bottle carousel.
HC-3000 HybriCycler Hybridization Oven Operation HybriCycler functions: • The “Power” switch turns the HybriCycler ON or OFF. • The “Motor” switch turns the bottle rotation ON or OFF. • The “Temperature Control” displays and controls the temperature setting within the hybridization chamber. 1. To operate the unit, press the “Power” switch to the ON position. 2.
HC-3000 HybriCycler Hybridization Oven 6 3. Press the “Motor” switch to the ON position. Setting the Temperature Controls Whenever power is applied to the controller, the software revision number is displayed for several seconds. While the software revision number is being displayed, the intensity of the display digits alternates between full and half brightness, and on some models the two outside decimals points blink on and off.
HC-3000 HybriCycler Hybridization Oven 7 Care and Cleaning The drawer-style spill tray is designed to contain any spills that leak from any of the bottles within the Hybridization Chamber. The spill tray has a fluid capacity of 300ml. The inside of the HybriCycler Hybridization Chamber should be cleaned with a damp cloth. The bottle carousel and outside of the HybriCycler can be cleaned with soap and water.
HC-3000 HybriCycler Hybridization Oven 8 Hybridization Techniques There are really two main steps to a hybridization reaction: hybridizing two strands of complimentary DNA and detection of the hybridized DNA. Nucleic acid hybridization is a mechanism where strands of DNA in a single stranded state have their complements bind together. The proximity of the DNA strands to each other determines the frequency of the binding events and is in fact successful binding is proportional to their concentration.
HC-3000 HybriCycler Hybridization Oven 9 1: Concentration of Species Target: How much target molecule depends on the species you expect to find. Cellular constituents may be expressed in large or small amounts; the trick is to start with enough target (~25 µg) and determine experimentally. Probe: plan to have more probe than expected target.
HC-3000 HybriCycler Hybridization Oven Situation G+C = 45-55% G+C < 45% G+C > 55% Evidence of probe target mismatching Response follow normal protocol Lower salt and temperature Raise salt and temperature Lower salt and temperature Target and probe is degraded on aqueous solution Hybridize in a formamide-based buffer Unacceptable high background Use less probe 10 Hybridize at lower salt/ higher temperature Wash with lower salt higher temperature Incubate with very low salt/change nuclease(solution) U
HC-3000 HybriCycler Hybridization Oven 11 Protocol 1: Random priming method for tagging DNA with fluorescein-lebeled nucleotide and others This method uses DNA polymerase to incorporate Fluorescene-11- dUTP into double stranded DNA probes. This protocol can be used to incorporate any tagged nucleotides. Equipment Micropipettes and tips Boiling water bath 1.5 mL Microcentrifuge tubes Microcentrifuge Cap lock for Microcentrifuge tube Water bath set to 37°C Reagents Deionized, sterile water EDTA, 0.
HC-3000 HybriCycler Hybridization Oven 12 12. 1 mL DNA polymerase 13. Mix gently and incubate at 37°C for 1 hour 14. Stop the reaction by adding 2 mL EDTA 15. Store probes at -20°C in the dark. Protocol 2: Hybridization to Nylon or Nitrocellulose Hybridization to nylon or nitrocellulose membranes containing Nucleic Acid is accomplished by adding single-stranded probe to the membranes that have been previously incubated with prehybridization solution.
HC-3000 HybriCycler Hybridization Oven 13 5. Denature the probe by placing the samples in the boiling water bath and heating for 10 minutes. Immediately transfer the tube to ice for 5 minutes (to prevent renaturation). Add 5 mL of hybridization buffer to the probe and transfer to the hybridization bottle containing the blot: AVOID pouring the probe directly onto the blot. 6. Incubate in the UVP HybriCycler, Hybridizer, or Minidizer 6 to 8 hours at 42 to 56 degrees.
HC-3000 HybriCycler Hybridization Oven 14 6. Tape the blot to a cardboard backing. 7. Cover with plastic wrap to prevent the blots from sticking to the x-ray film. 8. Place the cardboard containing the blots into the X-ray film folder. 9. In the darkroom, place a piece of X-ray film over the blot(s). 10. Place an intensifying screen on top of the film. 11. Close the film folder and clamp it. 12. Store at –70°C. The low temperature reduces light scattering and increases the length of exposure time.
HC-3000 HybriCycler Hybridization Oven 15 Product Warranty UVP’s quality HC-3000 HybriCycler System is guaranteed to be free of defects in materials, workmanship and manufacture for one (1) year from the date of purchase. Consumable and disposable parts including, but not limited to bottles, tubes and filters, are guaranteed to be free from defects in manufacture and materials for ninety (90) days from date of purchase.
