MagnaMedics. Move to Simplicity. HowTo: Automate MagSi-NGSPREP on a PrimaDiag workstation

Transcription

1 HowTo: Automate on a PrimaDiag workstation

2 automation protocol for PrimaDiag This HowTo describes the preparations needed for running automated protocols with MagSi-NGS PREP on PrimaDiag liquid handling platforms. Magnetic bead based offers an efficient solution for clean-up and size selection in NGS library preparation. The simple and flexible protocols can be adjusted to your specific application and NGS platform. All protocols described in the product manual have been automated for the PrimaMPS 6 and PrimaRWS 6 liquid handling platform and are available for download. Protocols for other system configurations are available on request - please contact our technical support. is For automation one of the following platforms are required and available at your PrimaDiag distributor: PrimaMPS 6 Product Discription Quantity Catalog number RWS 6 base unit Platform 0 x 60 cm allows or Z-axis 00 5 SBS deck 6 MPS 5 standard positions deck for PrimaMPS Z-axis Z-axis able to embed a pipetting head 0300 P8V50 8-channel pipeting head -50µl (position Z) 085 PCII full x PrimaControllerII license with the control computer 3000 TipsHolder50 Holder for tipbox 50µl Magnetic Working Unit Active magnetic unit for beads technology PrimaRWS 6 Product Discription Quantity Catalog number RWS 6 base unit Platform 0 x 60 cm allows or Z-axis 00 6 SBS deck 6 6 standard positions deck for PrimaRWS Z-axis Z-axis able to embed a pipetting head 0300 P8V50 8-channel pipeting head -50µl (position Z) 085 PCII full x PrimaControllerII license with the control computer 3000 TipsHolder50 Holder for tipbox 50µl HowToAutomateonPrimaDiag_v-0_09

3 Procedure: Step. Import your protocol The latest protocol files for your application are available on magnamedics.com/download/software All protocol files are compatible with PrimaController II v and above. Protocol number in manual Protocol Filename. Clean-up of enzymatic reaction MagSi-NGSprepCleanup_vX.X.zip. Left Side Size Selection MagSi-NGSprepLeftSideSizeSelection_vXX.zip.3 Right Side Size Selection MagSi-NGSprepRightSideSizeSelection_vX.X.zip. Double Sided Size Selection MagSi-NGSprepDoubleSidedSizeSelection_vX.X.zip X.X notes the latest version number. Open the downloaded file. When opening the file you might get prompted for a password. If the password is not known, please contact or your distributor. Copy the protocol file for the desired application to the computer running the PrimaController II software.. Open the PrimaController II software. 3. Click 'File' in the upper lefthand corner and choose 'Open project', followed by 'Browse'. Navigate to the application protocol file (.prws) and click 'Open'. To offer the user the flexibility required for different applications a number of variables have been implemented, including sample volume, sample positions and desired MagSi-NGS PREP bead ratio. Prior to running the protocols these variables are shown and can be set to user needs. 5. Click the check mark at the bottom of the screen 6. Edit the variables to your preferences. 7. When all variables have been set, click the refresh button. The amount of reagents needed for the protocol is recalculated and shown in the bottom part of the screen. Continue with the preperation of the deck. HowToAutomateonPrimaDiag_v-0_09

4 Protocol. Clean-up of enzymatic reactions. Left Side Size Selection.3 Right Side Size Selection. Double Sided Size Selection Variable Min Max Description Sample Volume (μl) 0 70 The volume of the samples First Column with First column of samples to be processed Last Column with Last column of samples to be processed Ratio : sample.0.8 Should be.8 for PCR cleanup. Alternatively, use the volume of beads recommended by your library prep kit manual. Elution Volume (μl) 35 - Volume of the final eluate Sample Volume (μl) The volume of the samples First Column with First column of samples to be processed Last Column with Last column of samples to be processed Left Side Ratio 0..5 The beads ratio corresponding with the desired left side cutoff size. Elution Volume (μl) 35 - Volume of the final eluate Sample Volume (μl) The volume of the samples First Column with 6 First column of samples to be processed Last Column with Last column of samples to be processed Right Side Ratio 0..5 The beads ratio corresponding with the desired right side cutoff size. Elution Volume (μl) 35 - Volume of the final eluate Sample Volume (μl) The volume of the samples First Column with First column of samples to be processed Last Column with Last column of samples to be processed Right Side Ratio 0..5 The beads ratio corresponding with the desired right side cutoff size. Left Side Ratio 0..5 The beads ratio corresponding with the desired left side cutoff size. Elution Volume (μl) 35 - Volume of the final eluate HowToAutomateonPrimaDiag_v-0_09 3

5 Step. Prepare the deck of your platform The protocols obtained from are defined with the following deck layout. If you want to use a different deck layout please contact the support team to request a protocol custom made for your needs.. Pipet tips. Pipet tips 3. Pipet tips. Pipet tips 5. Pipet tips * 6. Liquid waste position 7. Reagent position 8. Pipet tips * 9. Pipet tips * 0. Sample output position. Sample position (for PrimaRWS 6). Empty 3. Empty. Empty MWU. Sample position Positions marked with an asterisk are only applicable for the Left Side Size selection and clean-up protocols. A) Labware The following consumables are used in the predefined protocols. If you want to use different consumables please contact the support team to request a protocol custom made for your needs. Pipet tips 50 µl Microtiterplate Waste Position Consumable Cat. # Pipet tips Primadiag Tips50 Primadiag Tips50Refill Sample position Axygen 96 PCR well plate MM-Separator 96 SBS* PCR-96-FS-C MD90005 Reagent position Primadiag Reservoir 6030 Sample output position Thermo 96-Well Semi-Skirted AB-00 Liquid waste position Primadiag DPWmlsquare 6030 PrimaDiag Waste slider * Only applicable when using the PrimaRWS 6 platform HowToAutomateonPrimaDiag_v-0_09

6 B) Reagent position The reagents need to be present in the vertical slots according to the table below. Please note that the layout can be slightly different for a different protocol. It is not necessary to calculate the required volumes by hand, the software presents the outcome of these calculations prior to running the protocol. Protocol. Clean-up. Left Side Size Selection.3 Right Side Size Selection. Double Sided Size Selection Container content Container slot Total required volume (Sample volume (µl) *.8) * Number of samples + 00 µl Ethanol 70% 80 * Number of samples + 00 µl Ethanol 70% 3 80 * Number of samples + 00 µl Elution buffer Elution Volume (µl) * Number of samples + 00 µl (Sample volume (µl) * Ratio) * Number of samples + 00 µl Ethanol 85% 80 * Number of samples + 00 µl Ethanol 85% 3 80 * Number of samples + 00 µl Elution buffer Elution Volume (µl) * Number of samples + 00 µl (Sample volume (µl) *.8) * Number of samples + 00 µl Ethanol 85% 80 * Number of samples + 00 µl Elution buffer Elution Volume (µl) * Number of samples + 00 µl (Sample volume (µl) * Left Ratio) * Number of samples + 00 µl Ethanol 85% 80 * Number of samples + 00 µl Elution buffer Elution Volume (µl)* Number of samples + 00 µl Note: the 00 µl aliquot mentioned in all formula's is to account for dead volumes HowToAutomateonPrimaDiag_v-0_09 5

7 C) Sample position: The sample plate to be processed is placed on the Magnetic Working Unit. To prevent the loss of reagents, the samples in the plate should be positioned in colums of 8. For the enzymatic clean-up a minimum volume of 0 µl is required. To enable reproducable size selection a minimum of 50 µl sample volume is required for the size selection protocols. For the Right Side Size Selection protocol and the Double Sided Size Selection protocol only column to 6 can be used for samples while using these protocols. Sample positions for Left Side Size selection and Enzymatic clean-up protocol. Sample positions for Right Side Size selection and Double Sided Size selection protocol. D) Sample output position: On the sample output position an empty plate must be placed. After the run this plate will contain the sample DNA. Please note that the well position of the samples will be the same as the well position of the samples in the input position. HowToAutomateonPrimaDiag_v-0_09 6

8 Step 3. Run the protocol When finished with the preperation of the deck, click 'Continue & update', followed by the Start button Your protocol is now being performed. The estimated handling times are stated in the table below. Time to complete protocol. Clean-up of enzymatic reaction 96 samples 8 samples 3 samples 6 samples < 65 min < 5 min < 35 min < 5 min N.A. < 50 min < 0 min < 30 min. Left Side Size Selection.3 Right Side Size Selection. Double Sided Size Selection When the protocol has been completed, the Primacontroller software displays a message, and the arm of the PrimaDiag platform moves to the homing position. If you have any questions or require further help, please contact at support@magnamedics.com for assistance to automate your individual laboratory workflow with Software or software protocols (e.g. for liquid handling systems) are provided under European Union Public Licence (EUPL v..). The Software may be changed or adopted under these regulation. The usage of the software protocols is only permitted in combination with the usage of Contractual Products. The software protocols offered by are provided "as is" and any expressed or implied warranties, including, but not limited to, the implied warranties of merchantability and fitness for a particular purpose are disclaimed. In no event shall, or any of her contributors be liable for any direct, indirect, incidental, special, exemplary, or consequential damages (including, but not limited to, procurement of substitute goods or services; loss of use, data, or profits; or business interruption) however caused and on any theory of liability, whether in contract, strict liability, or tort (including negligence or otherwise) arising in any way out of the use of this software, even if advised of the possibility of such damage. HowToAutomateonPrimaDiag_v-0_09 7

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