USDA Sequenom MassArray iPlex Gold Protocol, Dec 19, 2012St. Amand, Page 1 of 8
Passwords
Workstation and DB server:
ID: administrator
PW: biomass
Typer Analyzer Software:
ID: charles
PW: darwin
Nanodispenser:
ID:operator
PW:operator
Shutdown Procedure
1. On the workstation computer:
a. Desktop>"Stop RT Processes"
b. Shutdown
2. On the Database computer, just shutdown.
3. Use the large power switch on the back of the MA instrument to power down.
Reboot Procedure
1. Use the large power switch on the back of the MA instrument to power up. The MA will require 24 to 48 hours to draw down the vacuum before the instrument is ready to use.
2. Reboot the database server:
3. After rebooting the workstation computer:
a. Log in
b. Desktop>"Start RT Processes"
c. Desktop>"Typer"
d. If you will not use the instrument right away, turn OFF the "High Voltage" button in the "Spectro Acquire" software.
iPlex Gold Protocol
Design Primers
1. Install the Assay Design software on any computer or use the Assay Design software on the MA workstation.
2. SNPs and short indel assays for the MA can not be designed unless the user has 30 to 300 base pairs of sequence information on BOTH sides of the SNP or indel.
3. The process of designing primers for the MA is complicated and the user MUST read the MassARRAYAssay Design 4.0 Software User’s Guide available on the MA workstation or from Paul.
Order Primers
1. Arrange the 1st-PCR primers in the order plate so that the forward primers are in one column and the reverse primers are in another matching column so that an 8-chanel pipetter may be used for dilution and pooling. The smallest synthesis scale is usually sufficient for 60 or more 384-well plates. The cost is about $300/assay set. NOTE: Capture primers for multi-SNPs are the SAME, no need to order both.
2. Arrange the extension primers in the order plate by mass from lightest to heaviest. Order the large, 200 nmol synthesis scale. The large 200 nmol scale will only be enough primer for two 384-well plates. The cost is about $200/assay set. You may want to order more wells of the larger mass primers.
Dilute Primers
1. Dilute 1st-PCR primersto 100uM each in NanoPure water ONLY.
2. Dilute extension primers to 500uM each in NanoPure water ONLY.
Pool1st-PCR Primers
1. Pool an aliquot of all 1st-PCR primersat 0.5uM each primer. 1000ul is sufficient for one 384 well plate. (5.0ul each primer, bring up to 1ml; or 200ul each primer, bring up to 40ml, aliquot into 2ml tubes). NOTE: Capture primers for multi-SNPs are the SAME, no need to pool both.
Pool Extend Primers (table based on 1 to 6x, may need to go to 4x)
1. Pool and test an aliquot of the extension primersprior to use. There are 2 ways to do this. Option 1, is to pool a small aliquot of the extension primers together using the worksheet below as a guide, but reduce the listed volumes by 10X. After testing, this original pool is discarded and a new pool (with modified volumes based on preliminary testing) is created for actual use. Option 2, is to pool the extension primers together using the worksheet volumes below. After preliminary testing, this original pool is modified by adding additional primer, for those low primers, before actual use.
Test the Extension Primer Pool
1. Turn on the MassArray unit 24 to 48 hours prior to use in order to have the vacuum system ready.
2. Check waste and supply water levels in the nanodispenser. Empty and fill if needed.Perform the weekly and daily nanodispensercleanings if needed (see page 7 for details).Remove the foil-wrapped primer testing chip from the 4°C cooler and equilibrate at room temperature for 30 min.
3. You will need the excel file generated during primer design (Name.xls). Do NOT edit this file. Put the file on Dolly and Paul will copy it to the workstation.
4. Combine4 ul of the extension primer pool and 96 ul of water. Put 23 ul in 4 wells ofthe primer testing plate. Mark the plate bottom for the well locations used in order to keep track of used chip positions. Note the chip barcode ID (60664781). Using the "Maintenance Menu", switch the nanodispenser insert and cover to the single pin (follow on-screen prompts). No need to resin-clean the primer pool. No need for calibration checks on the chip. Map and spot 4 reps on thechip using the nanodispenser (choose analyte only, no calibrant, volume check, & max dispense seed) and load the chip on the Mass Spec.You MUST have 5 nl to 12 nl per spot for good results (9 nl is the optimum). Use the left hand chip position for chip 1 and the right hand position for chip 2.
5. Using the "Assay Editor" application on the workstation, create a new customer, or projectas needed. Import ONLY assay group info from the excel file into your project (Deselect other 2 boxes). Edit assay info here if needed (especially allele calls). If the file fails to import, 1 or more of the allele call names is too long. Edit the "Name.xls" file using the NOTEPAD app (the file is really a text-only file).DO NOT USE THE POUND SYMBOL, COMMAS, SPACES, SLASHES, PERIODS, OR OTHER UNUSUAL CHARACTERS IN SAMPLE NAMES, ASSAY NAMES, FILE NAMES, PLATE NAMES, PROJECT NAMES, OR EXPERIMENT NAMES. IF YOU DO, YOU WILL HAVE PROBLEMS WITH THE SOFTWARE!
6. Using the "Plate Editor" application on the workstation, create a new Customer, Project, & Plate as needed. From the "Sample" tab, create a new sample project and then a new sample group. While naming the sample group, click on the open folder icon to import a list of sample names from a plain txt file with ONLY sample names in column preference order. Assign sample namesto wells, THEN assign assay info to wells.DO NOT USE THE POUND SYMBOL, COMMAS, SPACES, SLASHES, PERIODS, OR OTHER UNUSUAL CHARACTERS IN SAMPLE NAMES, ASSAY NAMES, FILE NAMES, PLATE NAMES, PROJECT NAMES, OR EXPERIMENT NAMES. IF YOU DO, YOU WILL HAVE PROBLEMS WITH THE SOFTWARE!
7. Use the "Chip Linker" application to enter the chip barcode. Select "Piezodispenser"for single-pin assays. Choose "ADD" plates to chips and choose "Create".
8. In the "Spectro Acquire" application, enter the chip barcode ID in the "Auto run Setup" tab. Press the "Barcode Report" button. Make sure that the report shows green and "Found" for each chip.
9. Turn OFF the "Use Calibrant" option and turn ON the "High Voltage" button and switch to the "Autorun" tab. Press "Start Autorun" and the instrument will read the chip barcode and begin collecting data.
10. While the data is being collected, switch the Nanodispenser insert back to the 24 pin insert.
11. Once the data has been collected, use the "Type Analyzer" application to examine the data. Find the correct Customer, Project, Plate, and Chip. Right-click on the chip and add the chip. Use the checkbox to select the chip and then your data will appear.
12. Check the peak heights from the extension primer pool test. You can select the wells of the plate and export the "Plate Data" to an XML file. You can use Excel to import the XML file, choose the XML-list file type upon import. All peaks should be of uniform height and be at least 10X greater than the background peak heights. Heights of 20 to 60 are good. If individual peaks are too low, estimate the percent increase needed and re-pool the primers with the new amounts or add additional primer to your pool as needed for low primer peaks.
1st-PCR
1. Make the iPlex Pre-Amp master mix as below. Do NOT use Tween-20 in PCRs.Vortex and centrifuge.
2. Add 3ul MM to a RT-384 well plate (T-3157-1 ONLY). Do NOT skip rows or columns, or you will have to move samples manually at the chip spotting stage. If you less than a full 384 well plate, plan to NOT have blanks. Centrifuge.
3. Add 2ul DNA to the 384 well plate. Seal the plate with a rubber mat. Vortex and centrifuge.
4. Thermocycle using the"iPlex-Pre"PCR program.
iPlex-Pre (Block profile, 2h 40min)
1. 95C, 2 min
2. 95C, 30 sec
3. 56C, 30 sec
4. 72C, 60 sec
5. Goto step 2, 44 more times
6. 72C, 5 min
7. 10C, 5 min
SAP Cleanup
1. Dephosphorylate the remaining dNTPs using SAP.
2. Centrifuge the1st-PCR plate.
3. Mix the SAP reagents as below.You may need Tween-20 to break the surface tension of the solution. Use only a tip-touch of Tween-20. Vortex and centrifuge reagents.
4. Add 2 ul of SAP MM to each well of the 1st-PCR plate (total vol. 7 ul). Seal the plate with a rubber mat. Vortex and centrifuge.
5. Thermocycle usingthe iPlex-SAP program.
iPlex-SAP (Block profile, 1 hour)
1. 37C, 40 min
2. 85C, 5 min
3. 10C, 5 min
iPlex-SBEExtension
1. Centrifuge the SAP treated plate.
2. Mix the iPlex-SBE extension MM as below. You may need Tween-20 to break the surface tension of the solution. Use only a tip-touch of Tween-20. Vortex and centrifuge reagents.
3. Add 2 ul of iPlex-SBE MM to each well of the SAP treated plate (total vol. 9 ul). Seal the plate with a rubber mat. Vortex and centrifuge.
4. Thermocycle using the iPlex-SBE program.
iPlex-SBE (Block profile, 3 hours & 40 minutes)
1. 94C, 30 sec
2. 94C, 30 sec
3. 52C, 5 sec
4. 80C, 5 sec
5. Goto step 3, 4 more times
6. Goto step 2, 39 more times
7. 72C, 3 min
8. 10C, 5 min
Resin Cleanup
1. Condition the nanodispenser using 1M NaOH using the "Weekly Condition" program (10 min.).
2. Clean the nanodispenser using 100% ethanol using the "Daily Clean" program (30 min.).
3.After the 30 min ethanol clean, drain the sonicator and fill the ETOH bottle with a 50% ethanol solution.
4.Centrifuge theSBEplate, add 16ul to 20ul(20ul may be best??) NanoPure water to each well in the SBE plate, cover with a mat and centrifuge at 5700 rpm for 1 min.
5.Spread resin onto a 6mg dimple plate and let dry for about 10 to 60 minutes.Do NOTtouch the resin with your hands (it won’t hurt you, it will hurt the resin.) Store resin at 4C.
6. Turn the SBE+water plate upside down and align to the dimple plate.Hold both plates and turn them over so resin will drop into wells.Tap the resin into the wells.
7. Seal reaction plate with film, centrifuge at 5700 rpm for 1 min., and place on the plate on the rotatorfor at least 15 minutes. Thirty minutes is better, more time is OK. While waiting, take chips from the Deli fridge and equilibrate at room temperature for 30 min (one used chip for volume check and a new chip for spotting samples).
8. Centrifuge the reaction plateat 5700 rpm for 5 minutes to pellet the resin.
9.Clean the dimple plate with NanoPure water, dry it and store it.
Spot Samples onto MA Chip
1. Using the "Maintenance Menu", switch the nanodispenser insert to 24-pins (follow on-screen prompts) and REMOVE the single-pin vacuum drying cover.
2. Insure that you do NOT have blank rows or columns in the plate, or you will have to move samples manually to get rid of blanks. Place plate on the nanodispenser.Add a USED chip to the chip holder/scout plate.
3. Set up a transfer to run and select VOLUME CHECK in the method. Start with the following options: Aspirate time=8 sec, Aspirate Speed=50mm/sec, Dispense Speed=35mm/sec. Increase humidity in the nanodispenser by placing an open beaker of hot water inside. Click the STEP button, instead of run.This will transfer the first set of 24 samples.
4. Click the volume icon at the top of the screen and view the volumes. Optimum volumeis9 nlper pad, but 5-12nl will work.If you need to add more, increase the speed.If you need less, decrease the speed.
5. Once the appropriate dispense speed as been decided, stop the run.
6. Load a NEW chip to the chip holder in the appropriate position and note the chip barcode ID.
7. Load 40uL of calibrant in the white reservoir.
8. Select "Analyte and Calibrant" in the method.
9. Step into the program and check the volumes. If the volume is fine, run the full program (click the play arrow).
10. When done, seal the SBEplate and store in -20C.
11. Pipette out remaining calibrant from the white reservoir and return to tube, store at 4°C.
Load Chip and Run MA
1. Load the chip on the Mass Spec. Use the left hand chip position for chip 1 and the right hand position for chip 2.
2. Using the "Plate Editor" application on the workstation, create a new Customer, Project, & Plate as needed.From the "Sample" tab, create a new sample project and then a new sample group. While naming the sample group, click on the open folder icon to import a list of sample names from a plain txt file with ONLY sample names in column preference order. Assign sample names and assays to wells.DO NOT USE COMMAS, SPACES, SLASHES, PERIODS, OR OTHER UNUSUAL CHARACTERS IN SAMPLE NAMES, ASSAY NAMES, FILE NAMES, PLATE NAMES, PROJECT NAMES, OR EXPERIMENT NAMES. IF YOU DO, YOU WILL HAVE PROBLEMS WITH THE SOFTWARE!
3. Use the "Chip Linker" application to enter the chip barcode. Select"Nanodispenser-R" for 24-pin assays. Choose "ADD" plates to chips and choose "Create".
4. In the "Spectro Acquire" application, enter the chip barcode ID in the "Autorun Setup" tab. Press the "Barcode Report" button. Make sure that the report shows green and "Found" for each chip.
5. Turn ON the "Use Calibrant" option and turn on the "High Voltage" button and switch to the "Autorun" tab. Press "Start Autorun" and the instrument will read the chip barcode and begin collecting data.
6. Once the data has been collected, use the "Typer Analyzer" application to examine the data. Find the correct Customer, Project, Plate, and Chip. Right-click on the chip and add the chip. Use the checkbox to select the chip and then your data will paper.
7. You can examine the data and generate reports using "Typer Analyzer". You can change calls for groups of samples manually by holding the "Shift" key and using the lasso tool.
Nanodispenser Maintenance
1. Check waste and supply water levels in the nanodispenser. Empty and fill with NanoPure water if needed.
2. Weekly cleaning:
•Fill wells A1 through D6 of a 384 well plate with 1M NaOH (30ul).
•Place the NaOH plate on the left plate holder and secure with hold-downs.
•On the maintenance screen, choose "Condition Weekly". This takes 10 minutes.
3. Daily cleaning:
•On the maintenance screen, choose "Sonicator Solution Drain".
•On the maintenance screen, choose "Sonicator Solution Fill".
• Open the door and fill the wash station with 100% ETOH.
•On the maintenance screen, choose "Clean Daily". This takes 30 minutes.
•On the maintenance screen, choose "Sonicator Solution Drain", to remove the 100% ETOH.
•On the maintenance screen, choose "Sonicator Solution Fill".
• Open the door and fill the wash station and supply bottle with 50% ETOH.
Nanodispenser Tips
Operation / Time / Speed / CommentsAspirate Time / 8 – 10 seconds / Gives the quill pins enough time to load properly. If you use the standard setting of 3 seconds, the pins do not always have time to load completely and can have more variability in spot size.
Aspirate Speed / 40 – 60 mm/sec / Faster speeds tend to strip more liquid from the pin tips. Going at 40 – 60 mm/sec has shown increase uniformity in spot sizes.
Dispense Time / 0.0 – 1.0 seconds / Standard setting of 0 works in most cases, however if you have sample that is under dispensing, you can increase the dwell time to .2 seconds. If you require more, you can go up to 1 second of dwell time.
Dispense Offset / 1 mm / Standard setting. Not very important as the springs on the pins absorb the 1 mm of travel.
Dispense Speed / 35 – 200 mm/sec / Below 35 mm/sec the speed has no effect. Above 200 mm/sec and the motors may begin to stall.
Calibrant Speed / 140 mm/sec / All Calibrant should be spotting at 140 mm/sec
Ideal Spot Size / 8 – 10 nl / RS1000 - Optimal spot size is between 8–10 nl. For GENII chips, 8-10 nl works best.