one pre-wetted fiber pad on the gray side of the cassette. Place a sheet of filter paper on the fiber pad. Place the equilibrated gel on the filter paper. the pre-wetted membrane on the gel. Place a piece of filter paper on the membrane. Place a fiber pad on the filter paper. Close the cassette firmly, being careful not to move the gel and filter paper sandwich. Lock the cassette closed with the latch. Place the assembled cassette into the module. Place the module into the tank with the black electrode to the top and left. Add a cooling unit to the tank. Fill the tank with buffer to the “blotting” mark on the tank. Add a stir bar to the tank and place the tank on top of a stir plate. Set the stirring speed as fast as possible without splashing. the lid on the tank, matching the electrode colors. PART B: RUNNING THE GEL Connect the electrical leads to a power supply. Set to an appropriate voltage and current and run the transfer. After starting, check the area around the electrodes to make sure tiny bubbles are forming. This is evidence of an electrical current in the buffer. Remove any air bubbles under the gel or membrane by rolling them out. Use a specialized roller or simply a glass test tube. Stirring the buffer will help maintain a cool and even buffer temperature and homogeneous ion concentration. The lid only fits on one way, so make sure the module is oriented correctly. The voltage, current, and time should be selected based on the type of buffer, gels, and analyte(s). One option for SDS-PAGE gels containing proteins in TG buffer with alcohol would be a one-hour transfer at 100 V and 250 mA.
Go back to the gas regulator. Look at the gauge on the left; it should register approximately 10 psi. If it is significantly above or below this value, CAREFULLY AND SLOWLY adjust the white valve until the pressure is between 9 and 11 psi. Allow the incubator to equilibrate to the set point temperature and pressure. The relative humidity inside the incubator will increase as the water from the reservoir evaporates into the warm air. No need to change any settings for humidity. After 10 minutes check the temperature and % CO2. If the temperature is too low, give the incubator some more time to equilibrate. Confirm the temperature using a certified thermometer or temperature probe. If the CO2 is too high or low, check the regulator gauge. The right gauge should display several thousand PSI while there is gas in the tank (this cannot be adjusted). The left gauge should be user-adjusted to around 10 psi. When the temperature and % CO2 have reached the set points, the humidity will also have equilibrated to the appropriate level and cells can be safely placed in the incubator. Only open the interior door when you need to add or remove items from the incubator. Try to coordinate these openings with other users to minimize the total number of openings per day. When you open the interior door quickly remove or replace your item and close the door; do not leave the interior door open for any reason if the incubator is on. PART B: MAINTENANCE IMPORTANT: the CO2 inside the tank is such high pressure that some of it is liquefied. As gas is consumed and leaves the tank, the liquid amount grows smaller; however, the same pressure will register on the regulator as long as there is liquid CO2 in the tank. What this means is that you will not observe a decrease in the pressure of gas in the tank UNTIL there is almost none left, then the pressure will rapidly decrease (within the same day or so). Therefore, it is important that the tank is monitored and a spare tank is kept on-site so they can be switched out quickly when the original tank empties.
menu(s). Click OK when finished with this Read step. Enter Delay, Temperature Set, Shake or more Read steps if desired. When you have finished designing your procedure, you may click Validate to make sure the order of your steps is logical (this is only useful when you have entered more than one step). OK when finished designing your procedure. Enter a plate ID on the next menu and add comments if desired. Click Read. The Save As dialog box will open; give your data set a unique name and click Save. The sample tray will open. Insert your plate with well A1 oriented towards the upper left corner. Click OK, the tray will close, and the reader will scan the plate. The tray will open when the procedure is finished. Remove the plate and manually close the tray by pressing the black eject button on the front panel of the instrument. A window with a schematic of the plate containing data will appear. You may need to choose the wavelength in the Data drop-down menu to see the data. To export data to Excel, click the Excel icon next to the Data drop-down menu. To read another plate with the same procedure, click the Add Plate icon on the toolbar. Follow the above directions, starting with entering a plate ID. PART B: MAINTENANCE Routine maintenance of the instrument should include cleaning the plate carrier to remove sample residue (an shake for 3-5 seconds, followed by a delay before reading. If you do not turn on the Tungsten lamp, the Xenon flash lamp will be used. The Xenon lamp does not need to warm up before use. No more than 350 µL should ever be added to a microplate for use in this instrument, due to the risk of spills.
From the home screen, the softkey at the bottom right labeled “Basic ATC.” Press the softkey labeled “Set nm.” Enter the desired wavelength using the numeric keypad and press Enter. Choose cuvettes appropriate for the wavelength. Make sure they are clean on the inside and outside and free from scratches or stains. Wearing gloves, fill one clean cuvette at least halfway with the solution to be used as a blank. Fill another cuvette at least halfway with the solution to be tested for absorbance. If there is a shortage of cuvettes, the blank can be measured first, then removed, and the blank solution replaced with the solution to be tested. Carefully wipe the optically clear sides of the cuvettes with a Kimwipe or lens paper to remove any drops of liquid, dust, or fingerprints. Some plastics are not and not be useful for UV absorbance readings. The blank solution should be identical to or as similar as possible to the solution in which the compound to be measured is suspended. This is usually water or an aqueous buffer. Orient the cuvette such that the optically sides are aligned with the light path. It is best to handle cuvettes by the optically opaque sides to avoid leaving any residue on the clear sides.
Open the sample compartment. Insert the blank cuvette in the sample holder labeled “B”. Orient the cuvette such that the optically clear sides are aligned with the light path. Insert the sample cuvette in the first sample holder, labeled “1”. If there are additional samples and enough cuvettes to hold them, insert these in the other four sample holders and note their positions. Close the sample compartment. Locate the numbered sample selection keys (arranged in a circle on the keypad). “B” to select the blank. the softkey labeled “Measure blank”. The display should read 0.000A. Press the sample selection key with the number of the sample you wish to analyze. The sample holder carousel will rotate and the instrument will analyze the selected sample, A 1/10 dilution is a good place to start. Use the blank solution to dilute the sample. If the absorbance is still too high, dilute again and repeat until an accurate measurement is obtained. Then, multiply the absorbance reading by the dilution factor to obtain the absorbance of the original concentration.
switch on the back of the instrument. Wait for the instrument to initialize and complete internal From the home screen, press the softkey at the bottom right labeled “Basic ATC.” Press the softkey labeled “Set nm.” Enter the desired wavelength using the numeric keypad and press Enter. Choose cuvettes appropriate for the wavelength. Make sure they are clean on the inside and outside and free from scratches or stains. Wearing gloves, fill one clean cuvette at least halfway with the solution to be used as a blank. Fill another cuvette at least halfway with the solution to be tested for absorbance. If there is a shortage of cuvettes, the blank can be measured first, then removed, and the blank solution replaced with the This option will allow the user to make basic absorbance measurements at a given wavelength. Some plastics are not and would not be useful for UV absorbance readings. The blank solution should be identical to or as similar as possible to the solution in which the compound to be measured is suspended. This is usually water or an aqueous buffer. It is best to handle cuvettes by the optically opaque sides to avoid leaving any residue on the clear sides.
Repeat 14-15 with all other samples. When you are finished, clean the cuvettes by first emptying them, then flushing with several changes of purified water. Next, flush with several changes of 70-95% ethanol. Finally, dry upside down on a paper towel before storing in a safe place. If you are the last to use the spectrophotometer, power off the instrument. PART B: DNA ANALYSIS BY SCANNING From the main menu, highlight “Nucleic Acid Tests” and press Enter. Select “DNA with Scan 260/230”. Parameters will display; press the softkey labeled “Run Test”. Insert a cuvette filled with the blank solution appropriate to your experiment into the sample holder. Press the softkey labeled “Collect Baseline” and allow contaminating residue. Disposable plastic cuvettes can be used a few times before they become scratched or stained and need to be discarded. Clean these carefully and they will last for several uses. Quartz cuvettes look like glass and are made to have a long life. Treat these with special care because they are very expensive. They will last many years if used carefully.
printout of your results. SOP SPC-003 NANODROP 1000 SPECTROPHOTOMETER Title: Operation of NanoDrop 1000 Spectrophotometer 1.0 SCOPE AND APPLICATION: This SOP outlines the policy and procedure for operation of the NanoDrop 1000 Spectrophotometer. 2.0 SUMMARY OF METHOD MATERIALS NanoDrop 1000 spectrophotometer Attached computer with ND1000 software Kimwipes, DI water, p2 pipette PART A: OPERATION Open the ND1000 software on the attached computer and select the analysis module of interest (nucleic acid, protein, etc). Follow prompts to initialize the spectrophotometer by pipetting 2 uL of DI onto the bottom pedestal and clicking OK. When this is finished, wipe the water off with a kimwipe. Establish a reference or blank using the appropriate buffer. Pipette 1-2 µL of blanking buffer onto the bottom IMPORTANT: do not handle the optic cable on the NanoDrop. This can damage it and cause problems with the instrument. When lifting and lowering the pedestal, handle only by the metal part at the bottom. Keep the instrument away from objects that could fall on or snag the cable to prevent damage.
PART A: SINGLE WAVELENGTH READINGS Turn on the spectrophotometer by pressing the power button. Wait for the instrument to initialize and complete internal tests. The default home page will be displayed. Press 1 on the number keypad to choose Single Wavelength. the wavelength you would like to use, then press the green ► button on the keypad to accept. Insert a cuvette containing the appropriate solution. Press the 0A/100%T button on the keypad to take a blank reading. Remove the blank cuvette and insert a cuvette containing the sample. Press the green ► button to take a reading. The absorbance will be displayed. If the absorbance value of a sample is above the range of the instrument, you instrument are optically The light path travels from right to left across the instrument. It is best to handle cuvettes by the optically opaque to avoid leaving any residue on the clear sides. The blank solution should be identical to or as similar as possible to the solution in which the compound to be measured is suspended. This is usually water or an aqueous buffer. A 1/10 dilution is a good place to start. Use the blank solution to dilute the
default home page. Enter the start and end wavelengths and press the green ► button. Insert a cuvette containing the appropriate solution. Press the 0A/100%T button on the keypad to take a blank reading. Remove the blank cuvette and insert a cuvette containing the sample. Press the green ► button to take a reading. The absorbance spectrum will be displayed. Repeat with any other samples. When you are finished, clean the cuvettes by first emptying them, then flushing with several changes of purified water. Next, flush with several changes of 70-95% ethanol. Finally, dry upside down on a paper towel before storing in a safe place. If you are the last to use the spectrophotometer, power off the instrument. these with special care because they are very expensive. They will last many years if used carefully. The blank solution should be identical to or as similar as possible to the solution in which the compound to be measured is suspended. This is usually water or an aqueous buffer. A 1/10 dilution is a good place to start. Use the blank solution to
PART C: KINETICS From the Standard Methods home screen, press 6 for Kinetics. Enter the wavelength, delay time (if any), duration time, and interval length using the numeric keypad and arrows. Press the green ► button to continue. Select the measurement mode using the left and right arrows (you will most likely want to select “Slope”). Press the down arrow to get to units, then enter the units manually or press the Options button to choose from a list of units. Press the down arrow and change the “Factor” setting if you need to multiply results by a dilution factor. Press the green ► button to continue. Insert a cuvette containing the reference sample and press the 0A/100%T button to take a blank reading. Insert a cuvette containing the sample and press dilute the sample. If the absorbance is still too high, dilute again and repeat until an accurate measurement is obtained. Then, multiply the absorbance reading by the dilution factor to obtain the absorbance of the original concentration. Never use any abrasive cleansers, soaps, or brushes on cuvettes. This can scratch them or leave a contaminating residue. Disposable plastic cuvettes can be used a few times before they become scratched or stained and need to be discarded. Clean these carefully and they will last for several uses. Quartz cuvettes look like glass and are made to have a long
MODES OF OPERATION If RoDi unit is in Run mode, you will see a number (such as “17.6”) on the digital display. This is the resistivity of the water in megaohms•cm and indicates the purity of the water. While in Run mode, water is being purified continuously. Unit should be in Run mode to dispense water. If RoDi unit is in Standby mode, you will see “Sby” on the LED readout. The unit should be left in Standby mode when not in use to conserve the UV bulb, RO membrane and cartridges. If RoDi unit is in Idle mode, you will see “IdL” on the LED readout. Idle mode halts purification until Start/Stop is pressed and unit enters Run mode. Idle mode should be avoided to ensure a supply of high purity water at all times. PROCEDURE: OPERATION Locate the container into which you intend to dispense purified water. Identify the operational mode of the unit by using the guidelines in the above section. If it is not in Run mode, press Start/Stop. A number indicating the resistivity of the water should appear. If necessary, allow the resistivity to rise until water has reached the desired purity. A resistivity of at least 16 megaohms•cm is adequate for most applications. Position the container under the valve without touching the valve to avoid contamination of the unit. Press down on the draw-off valve lever to dispense water. Lever will click down and stay in position. When finished, move the lever up to the off position. COMMENTS The more pure the water, the higher its resistivity will be. In Standby mode, the pump will operate for 10 minutes out of every hour, and the UV light will operate for 10 minutes out of every four hours. Glass is the best storage material for water containers, especially for long-term storage, because it does not leach materials like plastic does. Unit should be left on at all times. If unit is turned off, locate the power switch on the back of the unit and turn on. Notify the lab assistant if unit is turned off.
FLU-001 (Quantech Fluorometer) SOP removed. Equipment no longer in use in our labs. Evelyn Goss FLU-003 (NanoDrop 3300 Fluorospectrophotometer) SOP removed. Equipment no longer in use in our labs. Evelyn Goss PCR-002 (Bio-Rad iCycler for endpoint PCR) SOP removed. Equipment no longer in use in our labs. Evelyn Goss PCR-003 (Stratagene Mx4000) SOP removed. Equipment no longer in use in our labs. Evelyn Goss PCR-006 (Bio-Rad iCycler for Real-time qPCR) SOP removed. Equipment no longer in use in our labs. Evelyn Goss SPC-005 (GE Nanovue Spectrophotometer) SOP removed. Equipment no longer in use in our labs. Evelyn Goss SPC-007 (Bio-Rad SmartSpec) SOP removed. Equipment no longer in use in our labs. Evelyn Goss ELC-004 (Edvotek Electrophoresis Chambers SOP added. Equipment is frequently used in our labs. Evelyn Goss AUT-001 (VWR/Thermo Scientific Sterilemax Autoclave) Title of SOP and name of autoclave changed from “VWR” to “VWR/Thermo Scientific Sterilemax). Changed “tap water” to “deionized water.” The same model Sterilemax autoclave was sold by VWR and Thermo Scientific and we own one of each. Renamed to remove ambiguity. A support technician from Thermo suggested we use deionized water instead of tap. Evelyn Goss BAL-005 Scout Pro Balance) Corrected name of balance in the title from “Mettler-Toledo PL1502-S” to “Ohaus Scout Pro SP402.” Incorrect label was left from using MT balance SOP as a template. Evelyn Goss