From the Left Menu, click on "Molecular Biology" toolkit and then select DNA Assembly Reports.
​

You can click on your desired DNA Assembly reports and it will take you to the Assembly Report Record view as shown below.

The assembly report contains instructions, such as which materials are necessary for PCR reactions and assembly reactions, leading to the target construct.

(As a side note, ‘Export as CSV’ is helpful for users who would like to view the assembly report in a spreadsheet format.)

Click on ‘Link Sequences to Materials’. You should see a ‘Successfully linked sequences’ pop-up after materials are linked. Upon upload, sequences related to the assembly report were created. Linking to materials creates material equivalents of the related sequences.

Navigate to Inventory > Plates > Upload > DNA Plates. We will upload a plate containing the template for PCR. ‘DNA Plates’ is a generic plate upload for registering your plates into the system (as opposed to ‘Twist CSV’ and ‘IDT CSV’, which are uploads that expect the preferred formats of the DNA synthesis vendors). Click on ‘DNA Plates’. Click or drag ‘CombGoldenGate_PCRtemplate_plate.zip’ into the upload field.

Select ‘Generic 96 Well Plate’ as the plate type (by either scrolling or typing ‘96’ into the field to filter the list). Uncheck ‘Assign to Location’ (if you do not currently want to place the plate into a location of a refrigerator or freezer). Choose a lab, and submit.

This dialog simply informs the user that a barcode was not specified in the CSV of the uploaded plate. Click ‘Yes’ to proceed.

Double-click on the uploaded plate to enter the plate record view. Metadata about the plate, the display of plate wells, and a data table of aliquot information is shown. Double-click on an aliquot (either through the plate wells or through the data table) if you would like to enter an aliquot record view.

So far, we have added an assembly report and a plate containing a template for PCR. Now, we will start a workflow run. Go to Workflow > Runs > New Workflow Run. Name the workflow run, select the workflow definition ‘Demo Workflow PCR/Assembly’, choose a lab, and launch the workflow.

Through this workflow run, we will upload a plate of oligos from IDT, normalize the plate to a target concentration, generate assembly pieces through PCR, and use the assembly pieces in an assembly reaction. Click ‘Start Workflow’.

The Upload Plates tool (CSV) tool allows the user to upload plates from Twist or IDT, which are DNA synthesis vendors that ship plates with accompanying CSVs of a specific format. Click on ‘Start Task’.

For this example workflow we Select ‘IDT’.

Select ‘Generic 96 Well Plate’ as the plate type. Click or drag ‘CombGoldenGate_oligo_plate_IDTPlateUpload.csv’ into the upload field. Submit.

After completing a tool, a list of inputs and outputs is displayed on the tool summary page. In this case, we uploaded a plate of oligos, which can be viewed by clicking on the link. Click on ‘Go to Next Task’ to continue with the workflow.

The Normalization tool allows the user to normalize a plate to a target concentration or molarity across the wells. This tool calculates the necessary volumes, checks that the plate type’s wells can contain the final volume, and generates a liquid handler worklist for the volume transfers. Click on ‘Start Task’.

Select the oligo plate and proceed to the next step.

Input a target concentration or molarity. Uncheck ‘Transfer Before Normalization’. The option ‘Transfer Before Normalization’ allows the user either to normalize-in-place on the plate (i.e. generating one worklist of adding diluent directly to the plate), or to normalize on a new plate (i.e. generating two worklists of first transferring some volume to a new plate, then adding diluent to the new plate). Click ‘Next’.

Name the diluent container. This is necessary because liquid handler worklists expect a source plate of diluent (e.g. a reservoir plate of water). Click ‘Next’.

Name the normalization worklist and complete the tool.

Enter the Execute Worklist tool. This tool simply carries out the transfers contained in the worklist. Once the user generates a worklist and runs the worklist through a liquid handler, the Execute Worklist tool updates the source and destination plates in the system. Complete the tool.

Click on the output plate link and view the plate. The volumes and concentration of the wells have been updated. Proceed to the next task.

The PCR Planning and Inventory Check tool allows the user to choose which PCR reactions to proceed with, and then checks for which materials exist in inventory and which materials need to be ordered. Click ‘Start Task’.

In the first two steps, select an assembly report by clicking ‘Select DNA Assembly Report’, checking the box beside the ‘CombGoldenGate’ assembly report, and clicking ‘Select DNA Assembly Report’.

The user can choose to proceed with a subset of PCR reactions or all of them. For example, if the inventory check symbol (either a check mark or an exclamation point) indicated that some of the PCR products already exist in inventory, the user may no longer need to run all of the PCR reactions. In this workflow run, we do not have any of the PCR products in inventory, so we will leave all of the boxes checked, proceeding with all PCR reactions. Click ‘Next’.

In the next step, the user can select how the PCR reactions should be distributed across the plate. The first option uses Tm from the assembly report, which is calculated by Primer3. The second option assigns locations based on NEB’s preferred formulas for calculating Tm. If not using a zone gradient thermocycler, the user can choose the third option to disable zones based on Tm.

In the final step, select which aliquots you would like to use for the PCR reactions in this workflow run. If you have several aliquots of required materials across several plates, you can use ‘Fewest Plates’ or ‘Last Modified’ > ‘Automate Sample Selection’ to aid in the selection of which aliquots you would like to use. Name the output data and finish the tool. Click ‘Go To Next Task’.

The Run PCR tool generates a worklist to execute the PCR reactions and a PCR reaction map. A reaction map contains information about which materials (e.g. oligos, templates) will come together to create new materials (e.g. PCR products). Click ‘Start Task’.

The workflow definition is set up to automatically use the plate map and inventory material lists outputted by the upstream PCR Planning and Inventory Check tool. Because the inventory material lists contain information about which aliquots on which plates to use for all required PCR materials, using ‘Select Input Plates’ is unnecessary in this case. In other cases, ‘Select Input Plates’ allows for the selection of additional plates that contain aliquots of required materials for PCR.

The required materials for the PCR reactions are validated as in inventory. Click ‘Next’.

Input transfer volumes and name the PCR plate.

Name the PCR worklist and PCR reaction map. Click ‘Generate Worklist’. Proceed to the next task.

Complete the Execute Worklist tool, which updates the volumes of source and destination plates in the system.

Click on the link to the plate outputted by the Execute Worklist tool. The PCR plate is now updated with the PCR reactions.

The Construct Selection tool allows the user to proceed with a subset or all of the assembly reactions. Choose the ‘CombGoldenGate’ assembly report.

Assembly pieces to use can be selected through selecting/de-selecting checkboxes, through CSV upload, or through filtering by assembly pieces in inventory and selecting the available assembly pieces. In this case, keep everything selected and click ‘Confirm Selection’. Constructs that can be built based on the selected assembly pieces will be displayed. Name the output data and finish the tool.

The Assembly Reaction Planning tool generates a worklist for the assembly reactions and an assembly reaction map. The workflow definition is set up to automatically use the construct list outputted by the upstream Construct Selection tool.

The validation check verifies that all required assembly pieces for the assembly reaction are present.

If the user selects plates from which required materials for assembly reactions are missing, a validation error would be displayed listing the missing materials. This validation check ensures that the tool can generate a worklist to transfer volumes from various wells with all of the necessary materials for assembly.

Select ‘Generic 96 Well Plate’ as the plate type, name the assembly reaction plate, and either fill in individual volumes or check ‘Apply Universal Transfer’ to use the same volume across transfers.

Name the reaction map and worklist of the assembly reactions. Click ‘Generate Worklist’.

Click on the reaction map outputted by the Assembly Reaction Planning tool. The reaction map shows which materials combine to make a new material (e.g. an assembled construct).

Execute the assembly reaction worklist and view the output plate, which contains the target constructs.

Double-click on a well in the plate view on the upper right, or double click on a row in the data table at the bottom, in order to view an aliquot and its construct.

Click on the workflow run name to return to the workflow summary page, from there you can generate your "Workflow Report" by clicking "Generate Report"