SOP

Turning on the Jasco CD

  1. Turn on the nitrogen at the tank. (two turns of one valve) Check the flow rate to see that the top of the silver cylinder is at 20 SCFH.
  2. Enter the time you do this into the logbook.
  3. Turn on the computer and wait until it is totally booted up.
  4. If you'll be doing temperature work, turn on the chiller with the sliding black switch.
  5. Turn on the CD power switch. (rocking toggle, lefthand end of CD) Wait for the clicking noises to stop.
  6. Double click on the Spectra Manager icon to open that software.
  7. Choose Spectra Measurement and double-click it. The timer for 5 min. of nitrogen starts and then the lamp will light automatically. Check for its being lit. (If you just want to use the software and not the CD, click on the Cancel button. Skip would be used if the lamp has already been purged enough.)
  8. Open the shutter with a single click on its icon. (a yellow door will appear when it's open)
  9. Be sure the wavelength is set to 260-300 nm for watching the mdeg settle down. (If you need to change the wavelength manually, go to the nm (with 2 blue arrows) icon in the toolbar. Click it and adjust the wavelength there.)
  10. Watch the mdeg value on the lamp until it's stable. (a 0.4 mdeg range is good, takes about 20 min)
  11. Hint: Don't send your parameters to the CD before the lamp has stabilized. That changes what's displayed and it will be difficult to assess lamp stability.

Updated 4/10/2017

Setting up Spectra Measurement (for single scans at a single temp.)

  1. Under Spectra Manager, double-click on Spectra Measurement.
  2. Under the Measure button, pull down to Parameters.
  3. In the General window, choose the number of channels you want to collect and name them from the drop-downs.
  4. Choose a starting (high end) and ending (low end) wavelength.
  5. Select a data pitch (frequency). 0.5 nm is the default. 1 nm is for quicker scans.
  6. Start mode is Immediately unless your sample needs time in the darkness.
  7. Scan mode is Continuous.
  8. Scan speed: 50 nm/min is a good default. (correlates to the DIT)
  9. Set the CD scale to 200 mdeg/1.0 dOD for proteins and most other work. If you have small CD signal, set it at 20 mdeg/1.0 dOD.
  10. The FL scale setting is not applicable since we don't have that capability.
  11. Start with a DIT (Digital Integration Time) of 4 sec. (scan speed x DIT should be < 200)
  12. Set the bandwidth to be 1 nm as a good default.
  13. If you want the machine to read replicates, put a number in the Accumulation box. If you want the software to average those replicates, leave the Accumulation box checked. If you want to do the averaging yourself (and see all the replicates separately), uncheck the box.
  14. On the Cell Unit tab, enter the temp. and start condition you want, but no temp. control will occur.
  15. In the Control window, choose whether you want the machine to keep your baseline to subtract from your samples. Check the Shutter open/close automatically box. If your samples are light-sensitive, also check the Shutter closed between samples box.
  16. In the Information window, name your sample, its concentration, the solvent, whatever info you want to have in the file header. Check Display to be reminded to rename each new sample scan.
  17. In the Data window, check Auto save so it will always save your data and give it your folder name from the Browse button. (You can make a personal folder in the JASCO data folder on the desktop.) Choose a Format for this from the drop-down. Also check Send data to Spectra Analysis.
  18. To save all these parameters, click the Save button and give them a name so you can find them in your folder next time. This will be a .cdsp file.
  19. When you're ready to scan, click the OK button and your parameters will be sent to the CD.
  20. If you want the CD to keep your baseline to subtract from all your samples, click on B in the top toolbar. The temperature won't change until you start the first scan.
  21. Once you hit S to start the measurement, the window for how many cells you have will open. You won't be able to name the samples separately but will have to keep track yourself of which sample was in which cell.

Updated 4/25/2017

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Setting up Temperature/Wavelength Scan Measurement

  1. Close the spectra Manager software first.
  2. In Spectra Manager, double-click on Temperature/Wavelength Scan Measurement.
  3. Under the Measure button, pull down to Parameters.
  4. Click the Advanced Mode button at the bottom of the window.
  5. On the Tm setup tab, enter your starting and ending temperatures. Choose the Holder for Control sensor.  Choose a Data interval of 1.0 C. Choose the Temp. Gradient you want. (This dictates how fast your Tm scan will go.  Larger values make it go faster.) Enter a Wait time if desired and leave the Monitor sensor on Holder.
  6. Choose a start condition for the temp. control. Also choose whether you want the temp. to return to start (or another temp.) and whether you want the CD to Halt the ramping during the measurement. (recommended)
  7. On the Tm Param tab, choose how many data channels you want collected and the wavelength at which the Tm will be measured. The CD scale, D.I.T. and Bandwidth can be left as defaults.
  8. On the Spec Temp tab, enter all the temperatures where you want a scan taken. Counting the bottom temp. as 1, enter the number of scans this will be and click Apply. Uncheck Enable FL scan.
  9. On the CD Param tab, again choose the channels you want and enter the starting and ending wavelength for the scans. Data pitch is the frequency of measurements. You want Continuous scanning mode and 50nm/min is a good scanning speed. Leave the CD scale as the default and change the DIT to 4 sec. Ignore the FL settings. A bandwidth of 1 nm is good.
  10. Skip the FL Param tab since we don't have fluorescence capability.
  11. On the Control tab, choose Blank if you want a blank read at room temp. If you want a blank read at your start temp., it will be your first "sample". Check off to have the shutter open/close.
  12. On the Information tab, enter as much info as you want. Leave the Display box unchecked as it won't be relevant.
  13. On the Data tab, check Auto save to have the data go to your folder and select your folder. Also check the box to Send data to Spectra/Interval Analysis. Save these parameters to your folder by clicking the Save button and name the method.
  14. When you're ready to scan, send the parameters to the CD by clicking OK.
  15. To start the readings, click on S in the top toolbar.

Updated 4/25/2017

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Setting up Variable Temperature scans (single wavelength)

  1. In Spectra Manager, double-click on Variable Temperature Measurement.
  2. Under the Measure button, pull down to Parameters.
  3. On the Temperature tab, enter your Start temperature. Then fill in the chart for the interval step in degrees, the target temp., the speed for the heating, and a wait time. Leave the Control and Monitor sensors on Holder.
  4. On the Start/End tab, choose the control you want before the reading is taken and the End condition. It's good to cool off your cuvette if it's gone up to 95°, for instance.
  5. On the General tab, choose the number of data channels you want and enter the wavelength for your scan. Leave the CD scale as the default unless your sample has small CD signal. The DIT of 1 sec and bandwidth or 1 nm are good defaults.
  6. On the Control tab, check whether you want the CD to measure a Blank for you. It will only do this at room temperature. If you want it done at another temp., check None and your blank will be your first "sample". Do check the box to have the shutter open and close automatically.
  7. On the Information tab, enter a sample name and any other info you want to record. Do check the box for Displaying this window before each measurement.
  8. On the Data tab, check the Auto save box and browse to find your folder. Also check the box to send your data to Spectra Analysis.
  9. To save these parameters, click the Save button and give them a name so you can find them in your folder next time. This will be a .cdsp file.
  10. When you're ready to scan, click the OK button and your parameters will be sent to the CD.
  11. If you have only 3 samples and you want baselines for them, let the CD take a baseline from all 6 cell positions or it doesn't think it has any baseline samples.
  12. To start the scan, click on the S in the top toolbar. A temp. window will appear and you can see how close the temp. is to the start temp. you set.
  13. You won't see a line showing up in your data panels until the CD has collected two points.

Updated 4/25/2017

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Converting data to a useful form

  1. Open Spectra Manager.
  2. Under Analysis, you'll see File Converter II. Double-click that to open it.
  3. Your Source files are always Std. Ver. 2. In the Destination field, choose the format you want from the dropdown there.
  4. Choose a Source folder by browsing for your main folder (in the Jasco Data folder on the desktop). If you're making your folder for the first time, also make a subfolder within it called Converted Data or something like that.
  5. Choose the Destination folder to be the subfolder you built for receiving converted data.
  6. Click the Convert button and it happens fast.
  7. Go check that the correct file type made it into your destination folder.

Updated 4/10/2017

For reference:

[θ]λ = θobs x 1/(10 lcn)
[θ] = molar ellipticity in deg cm2 dmol-1
θobs = observed ellipticity at λ
l = pathlength (cm)
c = concentration of peptide (M)
n = # of amino acids

Closing down the CD
  1. To be able to record the HT voltage at 300 nm:
    1. Change the wavelength manually to 300 nm. (Click on the nm (with 2 blue arrows) icon in the toolbar and adjust the wavelength there.)
    2. Open the shutter.
    3. Record the voltage value in the log book.
  2. Close all the Jasco software so the drivers can be terminated.
  3. Switch off the CD.
  4. Close the nitrogen tank. Record the time you do this in the logbook.
  5. Turn off the chiller if you used it.
  6. After you've sent your data to yourself, turn off the computer.

Updated: 4/10/2017

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