Operating Instructions. BIO RAD SmartSpec 3000

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Section 7 Operating Instructions

Note: It is Good Laboratory Practice (GLP) to use the Smart Spec 3000 spectrophotometer to measure absorbances in the range of 0.100 to 1.500 AU.

1. Choose an assay. SmartSpec 3000 has six different assay keys in the second row of buttons. When you press one of them, it signals SmartSpec 3000 to clear its memory of absorbance readings from the previous assay, to reset the sample number to 1, and to reset the dilution factor to 1. Before the data buffer is cleared, SmartSpec 3000 will offer to print any unprinted data. All of the assays collect absorbance data and some of the assays are designed to convert those readings into concentrations or other useful information. The user interface of each assay is described in detail in the next section.

The available assays are:

DNA/RNA. In this assay, SmartSpec 3000 will automatically collect absorbance readings at 260, 280 and 320 nm. If you choose to subtract the background at 320 nm

(in Setup), then the A320 reading is subtracted from the A260 and A280 readings before the A260 and A280 absorbances are displayed or used in any calculations of nucleic acid concentration or in calculation of the A260:A280 ratio.

Calculations. Nucleic acid concentrations of dsDNA, ssDNA and RNA are determined by multiplying the A260 absorbance reading by a mass/absorbtion conversion factor and by the dilution factor, or by multiplying the difference between the A260 and A320 by the conversion and dilution factors.

Concentration = A260 * Conversion Factor * Dilution Factor or

Concentration = (A260–A320) * Conversion Factor * Dilution Factor

SmartSpec 3000 is programmed with default conversion factors for dsDNA, ssDNA and RNA. These factors can be modified at any time.

Species dsDNA ssDNA

RNA

A260 of 1.000

50.0 µg/ml

37.0 µg/ml

40.0 µg/ml

DNA and RNA oligonucleotide concentrations are reported in two different ways: by mass (µg/ml) and moles (pmol/µl); you can toggle between the two values by pressing

Conc. In order to make these determinations, SmartSpec 3000 requires the molecular weight of the oligo and either the molar extinction coefficient or the mass/absorbance conversion factor (as shown in the table above for dsDNA, etc.). Frequently the molar extinction coefficient and/or the mass/absorbance conversion factor is provided when you order an oligonucleotide from a custom synthesis laboratory. However, if you don’t know this factor, SmartSpec 3000 can estimate them for you in one of three different ways. In order of increasing accuracy, by length of the oligo, by composition of the oligo, (i.e., how many A’s, C’s, G’s and T/U’s) or by the primary sequence of the oligo. See Section 11.2 for a complete explanation of the calculations.

Protein. The Protein function will automatically select the proper wavelength for the Bradford (595 nm), Lowry (750 nm), BCA (562 nm) or UV Protein (260, 280, and 320 mm) assays, or you can select a different wavelength for another assay. The

Bradford, Lowry and BCA are all well-characterized colorimetric assays for protein concentration. The UV protein assay can determine protein concentration in a nucleic acid background.

The SmartSpec 3000 Bradford, Lowry, BCA and Other Protein assays use standard curves created by measuring the absorbances of solutions of known concentration to determine the concentration of unknown samples. If you do not want to construct a new standard curve, you may recall one from the on-board memory. SmartSpec

3000 has the capability of storing up to ten standard curves in memory. The stored curves are numbered automatically from 0 to 9, or you can overwrite the number.

Stored curves may also be assigned alphabetic names using the Alpha function of the number pad.

You also have the option of declining construction or recall of a standard curve, but without the standard curve, absorbance readings cannot be converted to concentrations.

The UV Protein assay, developed to find protein concentration in a nucleic acid background, as in a crude cell extract, calculates protein concentration by the equation

Concentration = C1 * (A

280

– A

320

) – C2 * (A

260

– A

320

) where C1 and C2 are constants. If you select the UV Protein assay, SmartSpec 3000 will first show you the current values for C1 and C2 and ask you to accept or modify them. The preset values for C1 and C2 are 1.55 and 0.76 respectively.

Standard Curve Construction. SmartSpec 3000 will calculate a mean and standard deviation for each standard replicate group (if replicates are specified) and then use those data to fit a linear standard curve. The instrument will report the slope and intercept of the standard curve and the square of the correlation coefficient for the curve (r 2 ).

The correlation coefficient (r) provides an indication of how well the linear polynomial regression model fits the data. In effect, the correlation coefficient is the square root of the proportion of explained variation to total variation of the regression.

r = (explained variation / total variation) 1/2

The amount of explained variation increases as the goodness of fit improves. In a perfect fit, all of the variation is explained, the explained variation equals the total variation, and the ratio of the explained variation to total variation becomes 1.000.

Or, more simply, the closer the correlation coefficient is to 1.000, the better the fit of the regression, and the better the estimate of concentration.

Storing Standard Curves. As many as ten standard curves may be stored in

SmartSpec 3000’s on-board memory. These may be recalled for use in determining concentrations in subsequent assays. A new curve will be assigned a number from

0 to 9 by the firmware and you can then give it an optional name of up to eight characters in length.

Printing Standard Curves. If you choose to print a full report at the end of an assay, the standard curve will be printed along with the information about the curve.

This will include the concentrations and absorbances of the standard and the slope, intercept and coefficient of the curve.

You can choose to print the same information about a standard curve when it is first created or when it is recalled from memory.

Limitations of the methods. The linear regression, when fit properly, generally produces reliable results at the extremes of the range of the standard curve. Results calculated from mean absorbance data outside the range of the standard curve may be unreliable. (For the purposes of this discussion, the range of the standard curve is defined as the absorbance range between the highest and lowest mean absorbances of the standards used to form the standard curve.) Calculated results that are determined from mean absorbances outside the range of the standard curve are extrapolations, and should be treated as such when determining the final results of your analysis.

The firmware will not be able to give a unique concentration for a given absorbance if the calibration data are not monotonic (i.e., two standards of differing concentration have the same absorbance reading).

Scan. SmartSpec 3000 can read any subset of the interval between 200 nm and 800 nm and then print the scan; the interval can be further subdivided into as many as four regions, and for each region the local minimum and maximum are identified. You may select a background wavelength and its absorbance will be subtracted from the absorbance at all other wavelengths in the interval of the scan. There are two scanning speeds: fast and slow. If you choose a slow scan, you will get the best data, but will take several minutes to complete, depending on the sample and the range being read. You may not specify more than one repetition for slow scanning. Scan data may be downloaded to an external computer via the serial port, or may be printed by SmartSpec 3000.

Kinetic. SmartSpec 3000 can collect absorbance readings at periodic intervals for a specified length of time so that you can collect kinetic data. You may specify a wavelength for background subtraction; background absorbances are collected at the same interval and are subtracted from the reading wavelength. You may then print out the data and/or download the data to another program for analysis. Permissible reading intervals range from 2 seconds to 999 seconds and data may be collected for as long as 9999 seconds.

OD600. This function is useful for monitoring growth of bacterial cultures.

SmartSpec 3000 can also convert the absorbance reading into a concentration with units of cells/ml if provided with a conversion factor. The conversion factor is multiplied by the absorbance reading and the dilution factor to estimate the concentration of cells in cells per ml. For example, if for certain E. coli an OD of 1.0

indicates a concentration of 5x10 8 cells/ml, then the conversion factor would be

5x10 8 . On the SmartSpec 3000 5x10 8 is entered in exponential notation using the

exp key (press 5, exp, 8 and Enter) and displayed as 5 e8. On the display, e refers to the base 10 and not to the base of the natural logarithm.

λλ

. The wavelength function allows you to choose up to three wavelengths for measurement and any other wavelength for optional background subtraction. It is a rapid and simple way to collect absorbance data without constructing standard curves or otherwise calculating concentrations.

2. Choose sample replicates. Samples may be read in replicate and you do not have to have the same number of replicates for each different sample. For each sample replicate group, SmartSpec 3000 will calculate a mean absorbance and standard deviation. When the full report is printed, each individual absorbance is reported along with the mean and standard deviation of the replicate group. If the default number of sample replicates has been set to 2–9 (in Setup), then SmartSpec 3000 will ask if any of the samples in this assay are to be read in replicate. The default answer is No.

Are any samples to be read in replicate? No:

• If you want to read samples in replicate, press Select to toggle from No to Yes and press Enter. You do not have to have the same number of replicates for each sample.

Enter number of sample replicates # 3

• Use the number keys to enter the number of sample replicates. The default number is the number chosen in Setup.

The default number of sample replicates can be changed during the course of the assay by choosing Options from the Ready screen.

3. Set the dilution factor. The dilution factor is reset to 1 each time an assay button is pushed. The dilution factor is always a number greater than or equal to 1; if one volume of sample has been diluted with nine volumes of buffer, the dilution factor is entered as 10.

Enter a dilution factor:

• Use the number keys to input the dilution factor and press Enter.

4. Zero the spectrophotometer. Zero (blank) SmartSpec 3000 by placing a cuvette containing the sample solution without analyte into the instrument and pressing Read

Blank. You can zero the instrument at any time during an assay.

5. Collect absorbance data. SmartSpec 3000 is now ready to collect absorbance data and convert them to concentration. Irrespective of whether you are going to read your samples in replicate or not, the Ready screen will be displayed.

Ready to Read Absorbance

<=Exit Assay >=Options

A. No sample replicates.

• Place your first sample in the cuvette chamber and press Read Sample. After the absorbance data are collected, they will be displayed as described below. At this point, you can put your second sample into the cuvette chamber and press Read

Sample.

• If you wish to begin reading samples in replicate, first return to the Ready screen by pressing Enter

Ready to Read Absorbance

<=Exit Assay >=Options and then press the right arrow key to choose Options.

<=Change next sample #

>=Change # of replicates

• Press the right arrow key to change the number of sample replicates.

Enter number of sample replicates: _____

B. Sample replicates.

• Place the first replicate of your first sample in the cuvette chamber and press Read

Sample. Before it collects the absorbance data, SmartSpec 3000 will ask for the number of replicates for the first sample.

Sample #1 Enter number of replicates: 3 >=Option

• Use the number keys to input the number of replicates and press Enter. The absorbance data for the first replicate will be collected and then SmartSpec 3000 will direct you to insert the second replicate

Insert Rep #2/3 Samp #1 and press Read Sample

This will continue until the last replicate of the first sample has been read. SmartSpec

3000 will then display the mean absorbance of all replicates of the first sample as described below. When the absorbance data are displayed, you can insert the first replicate of the second sample and press Read Sample.

• You can change the number of sample replicates at any time in the assay by first going to the Ready screen (if absorbance data are displayed, you can return to the

Ready screen by pressing Enter).

Ready to Read absorbance

<=Exit Assay >=Options

• Press the right arrow for Options.

<=Change next sample #

>=Change # of replicates

• Press the right arrow key to change the number of sample replicates.

Enter number of sample replicates: ______

• Use the number keys to input the new number of sample replicates and press Enter.

6. View the absorbance and concentration data. Absorbance data are automatically displayed after they are collected. In the case that you are reading samples in replicate groups, the mean of the replicate group is displayed. The standard deviation of the replicate group is available in the printed report.

A. DNA/RNA. Immediately after the sample is read, the data are displayed in the form

A280= x.xxx A320= x.xxx

Conc = xxx.xxx

µg/ml

• Press Abs to toggle the display to view the other absorbance data

A260= x.xxx Samp #1

Conc= xxx.xxx µg/ml

• Press A260:A280 to see the ratio displayed

A260= x.xxx A280= x.xxx

A260:A280 = x.xxxx

• For DNA oligos and RNA oligos, press Conc to see the concentration displayed in units of pmoles/µl.

A260= x.xxx Samp #1

Conc= xxx.xxx pmoles/µl

• To read the next sample, insert the cuvette in the chamber and press Read Sample.

From any of the above screens you can press Enter to return to the Ready screen.

Ready to Read absorbance

<=Exit Assay >=Options

B. Protein. Immediately after the sample is read, the data are displayed in the form

A595= x.xxx Samp #1

Conc= xxx.xxx µg/ml

• To read the next sample, insert the cuvette in the chamber and press Read Sample.

From any of the above screens you can press Enter to return to the Ready screen.

Ready to Read Absorbance

<=Exit Assay >=Options

The available options from the Ready screen are Change next sample number and Change number of sample replicates.

C. Scan. Immediately after the absorbances are calculated the screen will display the message

Press <Print> when ready

<=nm scale >=AU scale

• Press Print to begin printing the scan. If you do not want to print the scan, press

Cancel. From this screen, you can press the left arrow key to select nm scale and direct SmartSpec 3000 to print only a subset of the data collected.

Begin scanning at ____ nm

End scanning at ____ nm

You can also change the minimum and maximum values on the AU scale, by pressing the right arrow key.

Min AU scale:

Max AU scale:

• Enter the lower limit. If you want the lower limit to be a negative value, enter the absolute value of the lower limit and press Enter and then press Select. For example, if you want the lower limit to be -0.500, press the decimal key and the 5 key and then

Enter. The display will show

Min AU scale: +/- 0.500

Max AU scale:

• Now press Select and the negative sign will be displayed in front of 0.500.

Min AU scale: +/-0.500

Max AU scale:

• Press Enter again and now define the upper limit. The upper limit must be a positive number.

After the scan data are printed, or if you choose not to print the scan data, you are next offered the opportunity to download the data via the serial port to a computer.

Press <Enter> when ready to accept serial data.

• If you want to download the data to a computer via the serial port, make sure the proper connections are made and press Enter. If you do not want to download the data, press Cancel. After the data are downloaded, or after you press Cancel, the instrument will reset for the next scan and the Ready screen will be displayed.

Ready to Read Absorbance

<=Exit Assay >=Options

• Press the right arrow key to see the available options. You can change the number of repetitions (for fast scans), change the range, choose a background, define a subregion of interest and choose smoothing of the curve.

1. Repetitions 2.Range

3. Background >=More and

4. Regions 5. Smooth

<= Back, Cancel to return

• Press 4 to choose regions. You will be prompted for the number of regions and then the limits for each region. When the data are printed, the local minimum and maximum in each region are printed.

• Smoothing will take a running average of five consecutive data points, two on either side of the current wavelength, and plot those. For example, the absorbances at 455,

456, 457, 458, and 459 nm are averaged and plotted at 457 nm. Then the absorbances at 456, 457, 458, 459, and 460 are averaged and plotted at 458 nm.

Note: After the data are printed, you can go back to the Ready screen and turn on or off the smoothing feature and then plot the same data again. First go to the Ready screen, choose

Options, reset the Smoothing and then press Print.

D. Kinetics. Immediately after the data are collected, you will be asked if you want to print the data

Press <Print> for kinetics report.

• Press Print to print the report. If you do not wish to print the report, press Cancel. In either case, you will next be offered the chance to download the data into a computer via the serial port.

Press <Enter> when ready to accept serial data.

• If you want to download the data, make sure the connections are made to the computer and then press Enter. If you do not want to download the data, press

Cancel.

Ready to Read Absorbance

<=Exit Assay >=Options

After the data are downloaded SmartSpec 3000 will reset for the next kinetic data collection. From the Ready screen, you can change any of the parameters for the kinetic data collection.

E. OD600. Immediately after the absorbance data are collected, they are displayed along with the cellular concentration.

OD600 = x.xxx

Conc = xxxe xxx cell/ml

• You can read a second sample by placing the cuvette in the chamber and pressing

Read Sample. You can also return to the Ready screen by pressing Enter.

Ready to Read Absorbance

<=Exit Assay >=Options

Available options are Change next sample number and Change number of sample replicates.

λλ

. Immediately after data are collected they are displayed

A480=x.xxx

A580=x.xxx

A680=x.xxx

Samp #1

• You can read a second sample by placing the cuvette in the chamber and pressing

Read Sample. You can also return to the Ready screen by pressing Enter.

Ready to Read Absorbance

<=Exit Assay >=Options

Available options are Change next sample number and Change number of sample replicates.

7. Print the data. You can print data as they are collected, but it is more efficient to print absorbance and concentration data at the end of an assay. For assays other than Scan and

Kinetics, if you press Print, the display will show

1.Print unprinted Samp #s

2.Select # 3. Full Report

• Press 3 to print the full report which includes the absorbances and concentrations (if available) for each sample in the current assay.

• Press 1 to print all data collected since the last time you printed.

• press 2 to print only one sample

Enter Sample # ______ to print sample report.