Cary 300Bio easy User manual

Brugervejledning ©: Cary 300Bio User manual©: Cary 300Bio

final version 2015

©

Dr Anjana Sen

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**

Scan program

Turn on the airflow first before you even do anything, (remember to take off the tube-joints to avoid blast). Now join the tubes back together again. Turn on the computer and log in. Turn on the spectrophotometer. (Make sure the sample chamber is totally empty to begin with). Turn on the water-bath. Make sure that the fluid is circulating all the way properly, and there is no bubble in the circulating tubes (very important!). Leave it for 5 minutes. Open the Scan software program window. If it shows “Connect” at the top, you have to click the tab. If nothing happens, perform the following steps: Close the program window first. At the lower right corner of your computer desktop, on the taskbar, there is a tiny triangle. Click the triangle. It will open a small window with several icons. Right click on the “System Information” icon and click on “shut down”. Now open the “Scan” software program window again. This time it will show “Start” tab on top.

What to do if it still cannot connect: Open the System Information window. Click on the “Instrument” tab above. Select instrument type Cary 300. Click on the “Initialize” button at the lower left corner. Now open the “Scan” software program window again. This time it will show “Start” tab on top.

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Alternative Simply wait 5 to 10 minutes after you turn on the spectrophotometer and before you open any program window, so you will not see the “System Information is busy” message.

Wait until the initialization and calibration (lamp, filter, slits etc.) are finished.

Remember! When one program window is already open and you are opening one more program window, it will show the “Connect” button” instead of the “Start” button. Click on the “Connect” button in order to switch between program windows online. SETUP: GETTING READY When the “Start” tab is highlighted and bottom bar shows “idle” (ready to run), open the setup window by clicking on the “Setup” tab on the left-hand side of the Scan program window. Menus on the “Cary” tab: Choose X-mode and Y-mode. Select cycle mode if you want more than one scan. Choose options of cycle mode. In scan control menu, an ideal average time is 0.1 sec, an ideal data interval is 1.0nm and an ideal scan rate is 600nm/min. These values may vary after a new calibration. Do not alter these values frequently or abruptly. Select “Show status display” in the check-box. Keep “block” as temperature monitor for now.

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Menus on the “Options” tab: Advanced settings SBW/Energy: An ideal SBW is 2.0nm. However, it may vary depending on calibration. Do not alter this value abruptly. Beam mode: (1) If you are using samples (not more than 6) in the front cell holders and references in the rear ones, the beam mode will automatically choose to stay in the double beam mode. (2) If you are using more than 6 samples, the beam mode will automatically go to the dual single mode. (3) If you want to use rear cell holders for samples, the beam mode should be in the reverse mode. An ideal value of energy is 1.0. Source: If you are going to run the scan overnight or if you have to leave before the scan is finished, check the box for “Auto lamps off”. Select your required wavelength scan range between UV/Vis/UV-vis. A typical source changeover is 350nm. Signal-to-Noise: If you choose this option, leave the default values unchanged. Display option: Choose “Individual data”, if you want different scans in separate frames. Choose “Overlay data”, if you want all the scans in the same frame.

Menus on the “Baseline” tab: Choose baseline correction options, otherwise choose “none” if you do not want baseline correction.

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Menus in the “Accessories 1” tab: Cells: Always use cell changer. Select how many cells (samples) you are going to use by “Select Cells” checkbox. Use “Goto Cell” to move the sample chamber. Select 6×6 and multi-zero (and blank correction, if you need). Temperature: Select automatic temperature setting and temperature controller. Keep the block temperature as 25°C for now while you work on settings. You can change the block temperature to the required value for your experiment later on. Click the check-box for the block as temperature display for now. RBA: Not usually used. Skip Accessories 2 and 3 if you are not using auto sampler and sample transport.

Menus on the “Report” tab: Write all the necessary informations and descriptions in the report. Select graph options. Always choose to have peak labels. Select options in the “peak information” tab. Auto Store: Select storage on (prompt at start) to have a file name from the beginning.

Click the OK tab at the bottom of the setup window. Bottom bar will show that it is waiting for the selected temperature. When the selected temperature is reached (±0.5ºC), the “Start” tab will be highlighted again, that means the instrument is in idle status (ready to run).

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STARTING A MEASUREMENT When the temperature is absolutely critical: Now open the setup window one more time by clicking the “Setup” tab. Under “Cary” tab, change the Temperature Monitor option from block to probe 1 or probe 2. (Probe 2 is marked with black). Under “Accessories 1” tab, click the check-box for probe 1 or probe 2 on temperature display (where check-box for the block temperature was already on), so both the temperatures of the block and one of the probes will be displayed. Click “Goto Cell” and choose cell #12. The sample chamber will move forward to give access to all the cell holders. Fill cuvette #12 with double distilled deionized water (or the corresponding buffer used in the experiment), put it in the cell holder #12 and dip the selected probe into the cuvette. Make sure that the tip of the probe is totally immersed in the water. This will be your “dummy cuvette”. Click the OK tab at the bottom of the setup window. Bottom bar will show that it is waiting for the selected temperature. When the selected temperature is reached (±0.5ºC), the “Start” tab will be highlighted again, that means the instrument is in idle status. (Perform this following step only if temperature is absolutely critical. Otherwise skip this step.) Zeroing the instrument: Click the “Zero” tab on the left hand side of the Scan program window in order to zero the background absorption. A window will pop up with blank in all the selected cells. Keep it that way and click OK. Wait while the program brings background absorption values to zero in the selected cells one by one, which can be seen on the task bar below. Make sure that up to this point the sample chamber is totally empty except for the dummy cuvette and the lid of the sample chamber is properly closed at all time.

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Samples and references: Now fill the corresponding cuvettes with your sample and references, and close them with the white stoppers very delicately (not too tight or too loose). Put all your sample and reference cuvettes in the corresponding cell holders. Close the cell chamber-lid.

Set your desired temperature now: Under “Accessories 1” tab, in the temperature setup option, choose the desired temperature, click ok. Wait for 1 minute for temperature equilibration, which can be seen on the task bar below. When the selected temperature is reached (±0.5ºC), the “Start” tab will be highlighted again, that means the instrument is in idle status (ready to run).

Click the “Start” tab. Write the file name and sample names in the pop-up window.

Curves and graphs: When the scan is finished, use cursor mode (remember to change the free mode to the track mode) from the row of menus above in the Scan program window in order to find X-Y coordinates of particular positions on the absorption curves. Choose graph preferences/scale/text/maths etc tabs from the row of the menus above in the Scan program window, in order to edit the graphs and print.

Finishing up: Use “Goto Cell” button to bring all cell holders forward so all the cuvettes can be taken out. Close the program window. Turn off the water bath first, then turn off the Spectrophotometer and turn off the airflow.

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**OBS!!! Never do anything when the status bar is NOT saying “ready” or “done”.

Thermal program** Turn on the airflow, (remember to take off the tube-joints to avoid blast). Now join the tubes back together again. Turn on the computer and log in. Turn on the spectrophotometer. (Make sure the sample chamber is totally empty to begin with). Turn on the water-bath. Make sure that the fluid is circulating all the way properly, and make sure that there is no bubble in the circulating tubes (very important!). Leave it for 5 minutes. Open the Thermal software program window. If it shows “Connect” at the top, you have to click the tab.

If nothing happens, perform the following steps: Close the program window first. At the lower right corner of your computer desktop, on the taskbar, there is a tiny triangle. Click the triangle. It will open a small window with several icons. Right click on the “System Information” icon and click on “shut down”. Now open the “Scan” software program window again. This time it will show “Start” tab on top.

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What to do if it still cannot connect: Open the System Information window. Click on the “Instrument” tab above. Select instrument type Cary 300. Click on the “Initialize” button at the lower left corner. Now open the “Scan” software program window again. This time it will show “Start” tab on top.

Wait until the initialization and calibration (lamp, filter, slits etc.) are finished. Remember! When one program window is already open and you are opening one more program window, it will show the “Connect” button” instead of the “Start” button. Click on the “Connect” button in order to switch between program windows online.

SETUP: GETTING READY When the “Start” tab is highlighted and bottom bar shows “idle” (ready to run), open the setup window by clicking on the “Setup” tab on the left-hand side of the Thermal program window. Menus on the “Cary” tab: Choose required wavelength of the temperature run. A typical SBW value is 1.0nm and a typical value of “Average time” is 2.0sec. Choose Y-minimum and Y-maximum. A range of 0.0 to 3.0 is usually used. Choose start and return temperatures. Important tips! Keep both of them 25°C for now. You can choose the required temperature later on. If you choose simple collect, the number of stages is only one. If you choose advanced collect, you can choose desired number of stages. Click the check-box of “Collect Data”. Choose data interval (typically 1°C), rate (typically 1°C/min.), end temperature and hold time.

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Keep “block” as temperature monitor for now. Select “Show status display” in the check-box.

Menus on the “Options” tab: Collection options Source: If you are going to run the program overnight or if you have to leave before the temperature run is finished, check the box for “Auto lamps off”. Select your required wavelength range between UV/Vis/UV-vis. A typical source changeover is 350nm. Display option: Choose “Individual data”, if you want different curves in separate frames. Choose “Overlay data”, if you want all the curves in the same frame. Menus in the “Accessories” tab: Cells: Always use cell changer. In the “Select Cells” check-box, select how many cells (samples) you are going to use. Use “Goto Cell” to move the sample chamber. Select multi-zero. Blank Correction: (1) If your reference is the same for all the samples, you can fill cuvette #1 with your reference solution, put it in cell holder #1 and select “Blank Correction”. The program will automatically change the beam mode and automatically use cell #1 as reference for all. Click the “Blank Correction” check-box, in such a situation. (2) Otherwise use corresponding references for each of your individual samples. Leave the “Blank correction” check box unchecked in such a situation where you are using sample-reference pairs. The instrument will automatically select the appropriate beam mode. (But do not put any cuvette yet before working on settings is complete).

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Temperature Display: Click the check-box for the block as temperature display for now. RBA: Not usually used. Menus in the “Analyze” tab: Analysis Setup: Under analysis setup, smoothing is used for automatic smoothing if there are serious disturbances and fluctuations in the thermal curves. Keep it unchecked for now. Check “Derivative”, “Autocalculate” and “Hyperchromicity” check-boxes, if you wish the program to perform automatic calculations right after the collection is finished. If not, leave them unchecked for now.

Menus in the “Calculations” tab: Calculations and Corrections: Leave these functions inactive for now. These can be performed offline, when you are finished with your experiments.

Menus on the “Reports” tab Write detailed description of your experiments in comments and select report options, results, graph, user data form, parameters, etc. Always select for ASCII (csv), so you can have a list of the actual X-Y data for further plots and analyses.

Auto Store Select storage on (prompt at start) to have a file name from the beginning.

Click the OK tab at the bottom of the setup window. Bottom bar will show that it is waiting for the selected temperature.

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When the selected temperature is reached (±0.5ºC), the “Start” tab will be highlighted again, that means the instrument is in idle status (ready to run).

STARTING A MEASUREMENT When the temperature is absolutely critical: Now open the setup window one more time by clicking the “Setup” tab. Under “Cary” tab, change the Temperature Monitor option from the block to probe 1 or probe 2. (Probe 2 is marked with black). Under “Accessories” tab, click the check-box for probe 1 or probe 2 on the temperature display option (where check-box for the block temperature was already on), so both the temperatures of the block and one of the probes will be displayed. Click “Goto Cell” and choose cell #12. The sample chamber will move forward to give access to all the cell holders. Fill cuvette #12 with double distilled deionized water (or the corresponding buffer used in the experiment), put it in the cell holder #12 and dip the selected probe into the cuvette. Make sure that the tip of the probe is totally immersed in the water. This will be your “dummy cuvette”. Click the OK tab at the bottom of the setup window. Bottom bar will show that it is waiting for the selected temperature. When the selected temperature is reached (±0.5ºC), the “Start” tab will be highlighted again, that means the instrument is in idle status (ready to run). (Perform this following step only if temperature is absolutely critical. Otherwise skip this step.) Zeroing the instrument: Click the “Zero” tab on the left hand side of the Thermal program window in order to zero the background absorption. A window will pop up with blank in all the selected cells. Keep it that way and click OK. Wait while the program brings background absorption values to zero in the selected cells one by one, which can be seen on the task bar below.

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Make sure that up to this point the sample chamber is totally empty except for the dummy cuvette and the lid of the sample chamber is properly closed at all time. Samples and references: Now fill the corresponding cuvettes with your sample and references, and close them with the white stoppers very delicately (not too tight or too loose). Put all your sample and reference cuvettes in the corresponding cell holders. Close the cell chamber-lid.

Important tips! Do the hybridization beforehand prior to the recording of the actual thermal melting/denaturation Set your desired temperature now: (1) If you want to record a thermal scan of your hybridization: Under “Cary” tab, in the collect temperature option, choose the desired start and end temperatures, and click ok. Wait for 1 minute for temperature equilibration, which can be seen on the task bar below. When the selected temperature is reached (±0.5ºC), the “Start” tab will be highlighted again, that means the instrument is in idle status (ready to run). (2) If you prefer to perform hybridization without recording a thermal scan: Simply click the “Set Temp” button on your left hand side of the program window. Wait for 1 minute for temperature equilibration, which can be seen on the task bar below. When the selected temperature is reached (±0.5ºC), the “Start” tab will be highlighted again, that means the instrument is in idle status (ready to run). First up, then down. Finally the program with bring it to your desired start temperature. Click the “Start” tab. Write the file name and sample names in the pop-up window.

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Curves and graphs: When the temperature run is finished, use cursor mode (remember to change the free mode to the track mode) from the row of menus above in the Thermal program window in order to find X-Y coordinates of particular positions on the thermal curves. Choose graph preferences/scale/text/maths etc tabs from the row of the menus above in the Thermal program window in order to edit the graphs or print.

Finishing up: Use “Goto Cell” button to bring all cell holders forward so all the cuvettes can be taken out. Close the program window. Turn off the water bath first, then turn off the Spectrophotometer and turn off the airflow. **

OBS!!!

Never do anything when the status bar is NOT saying “ready” or “done”.

Analyses and Calculations offline Menus in the “Analyze” tab: Analysis Setup: Under analysis setup, “Smoothing” is used for automatic smoothing if there are serious disturbances and fluctuations in the thermal curves. Typical value for interval is 1.0 and typical value for filter size is 5.0. Check “Derivative” and “Autocalculate”. Typical value for interval is 1.0. Important tips! An appropriate value for filter size should be 19, in order to locate the correct and unique monophasic transition, (though the default values shows 5, which would otherwise point at all small bumps in the curve).

Typical values for low and high calculation limits are −10 and 110, respectively. Select the “Hyperchromicity” check-box, select van’t Hoff calculations and all the details. For example, select non-self-complementary for duplexes and self-

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complementary for hairpins. Define the values of strand concentration, molecularity (= 2 for double helix) and temperature for DeltaG calculation (which is typically 37°C in our cases).

Menus in the “Calculations” tab: Calculations and Corrections: Choose the appropriate equation for your experiment. Write the correction temperature and normalizing options.

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types of cuvettes UV range (280 - 400 nm)

quartz crystal

visible range (400 - 750 nm)

plastic (disposable), glass

glass

amorphous, 80% silicone dioxide

quartz

crystalline lattice, 99% silicone dioxide

plastic cuvette

glass cuvette

quartz cuvette

may get damaged by organic solvents

does not

does not

absorbs UV light

does not

does not

use and throw, fast assay

better accuracy

high performance, high accuracy

visible range

UV-Vis

UV - NIR

cheap and handy

fragile

fragile and expensive

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difference between cuvettes for absorption

cuvettes for fluorescence

only 2 transparent sides

4 transparent sides

2 mat / black / frosted sides

no mat / black / frosted sides

because, emitted fluorescence is because, light-beam travels measured at 90° angle w.r.t. the straight into the detector excitation light-beam

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quartz cuvette washing instructions step 1: Use soft narrow long-tip plastic pipettes to take out sample from the quartz cuvettes, so you don’t make scratches on the inside walls of the cuvettes.

step 2: Make 2% vol./vol. solution of Hellmanex III in deionized double distilled water (milliQ).

step 3: Immerse the quartz cuvettes in this solution for minimum 30 minutes. Remember to wear gloves.

step 4: Put on gloves. Take out the cuvettes from Hellmanex solution and wash them under running single distilled water. Hold each cuvette by fingers, fill them under running single distilled water, then hold them upside down and carefully shake them empty. Repeat this 40 - 50 times for each cuvette separately, until the soap is completely washed out, to make sure that, no remnant of the soap is left any more in the end.

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Be very careful, because the soap is slippery, and the cuvettes are expensive. step 5: Now wash each of them in deionized double distilled water (milliQ) minimum 10 times, the same way as in step 4, to make sure that, no remnant of the single distilled water remains behind.

step 6: Repeat step 5 with technical grade Ethanol 5 times each, to get rid of water and eventual water marks.

step 7: Repeat step 6 with technical grade Acetone 5 times each, to get rid of alcohol and also to accelerate drying process.

step 8: Pass dry air-flow (pre-dehumidified by CaCl2) through each Acetone-washed cuvette to dry them up, or leave them overnight for automatic drying.