Assembly design

1. To design primers for the pre-assembly PCRs, navigate to the NEBuilder Assembly Tool.

2. Click Settings on the main page.

   

   NEBuilder main page

3. Ensure the settings are as expected and that the PCR polymerase/kit is set to what you plan to use — we typically use Q5. Click Done.

   

   NEBuilder settings

4. Click NEW FRAGMENT.

   • From Benchling, paste in the sequence of the ENTIRE vector backbone you plan to clone into, even if you will be performing a double digest to open it.
   • Give the sequence a name, click PROCESS TEXT, and check VECTOR and CIRCULAR.
   • Select how you will be linearizing the backbone. If you choose Restriction Digest, select the 5′ and 3′ restriction sites you will be using.
   • Click Add.

   

   Adding the vector backbone

5. Click NEW FRAGMENT again. You will likely be generating the rest of your fragments via PCR — if so, paste in only the sequence to be amplified. Give the sequence a name, check "Autogenerate primers," and click Add.

6. Repeat step 5 for as many fragments as necessary. When finished, the Build page should look something like this:

   

   Completed assembly build

7. You can now choose to add spacers or cut sites between each fragment. Click the pencil (edit) icon on the backbone vector — pPD95.75 in this example — and select whether to regenerate the restriction sites. Generally "Min" is the best choice, but be careful not to change the reading frame if you are assembling a fusion protein.

   

   Site regeneration options

8. Repeat step 7 if you want to add spacers between other fragments, or generate new cut sites between them.

9. Click Done to get the final assembly. Copy the "Assembled Color-coded Sequence" and import it into Benchling for markup and annotation. Ensure the sequence is what you expect, and that the junctions between fragments aren't generating new cut sites or abolishing cut sites you wanted to retain.

10. If all is satisfactory, order the designed oligos. Be sure to record the 3′ Tm and 3′ Ta temperatures — the 3′ Ta will be used for the PCRs that generate the fragments.

Assembly reactions

1. Before assembly, generate all the fragments to be assembled, whether by synthesis, restriction digest of a plasmid, or PCR.

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   Restriction digest: confirm the digest completed by running an aliquot on a gel. In our experience you do NOT need to gel purify the correct-sized band — the off-target band will not impede the assembly. However, you DO need to inactivate the restriction enzymes. Some NEB enzymes can be heat inactivated at 65°C for 20 minutes, but check the specifications for the enzymes you are using. If you can't heat inactivate, purify using the PCR purification kit.

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   PCR: use a high-fidelity polymerase from NEB, typically Q5. The polymerase you choose should match the one you selected in the NEBuilder Assembly Tool. Run 25 μL reactions for 35 cycles, using the 3′ Ta the Assembly Tool provided. If the combined PCR products make up more than 1/5 of the final assembly reaction, purify the PCR reactions — in general, it's probably better to purify regardless.

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2. Thaw the reaction constituents on ice. Use the Qubit dsDNA BR kit to quantify every fragment.

3. Calculate the molar amount of each fragment to include. See the HiFi assembly manual for details, which change depending on the number of fragments being assembled. Use the NEB Calculator for mass-to-moles conversions.

   Example of two recent reactions

   Fragment	Ratio	Assembly #1	Assembly #2
   Vector backbone	50 ng	3500 bp (XbaI/EcoRI pPD95.75 fragment) = 0.023 pmol	3500 bp (XbaI/EcoRI pPD95.75 fragment) = 0.023 pmol
   Fragment 2	2:1 insert:vector molar ratio	0.046 pmol = 46.76 ng of 1645 bp cel-osm-9p	0.046 pmol = 89.23 ng of 3139 bp cel-tax-4p
   Fragment 3	2:1 insert:vector molar ratio	0.046 pmol = 79.79 ng of 2807 bp bma-osm-9	0.046 pmol = 44.43 ng of 1563 bp bma-tax-4
   Total	0.03–0.2 pmol	0.115 pmol	0.115 pmol

   Here we start with 50 ng of the backbone and calculate the number of pmol that represents, based on the size of the cut fragment being assembled. Multiply that amount (0.023 pmol) by two to get the 2:1 insert:vector ratio. 0.046 pmol of the 1645 bp cel-osm-9p is 46.76 ng.

   Using the calculated ng of each fragment and the measured concentration from the Qubit, calculate the volume to add for a final volume of 5 μL. This is less than the final volume given in the NEB manual, in order to save reagents — perform the calculations as recommended in the manual, then divide all the constituents by 4.

4. Assemble the final reaction in a PCR tube.

   Measured fragment concentrations

   Fragment	Concentration (ng/μL)
   XbaI/EcoRI pPD95.75	11.8
   cel-osm-9p	120
   bma-osm-9	117
   cel-tax-4p	95.4
   bma-tax-4	95.8

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   The product coming from the digestion will be much less concentrated than the product from the PCRs, unless you clean up the reaction and elute in a volume much smaller than 50 μL.

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   Final reaction volumes

   Fragment	Assembly #1	Assembly #2
   Vector backbone	50 / 11.8 / 4 = 1.06 μL XbaI/EcoRI pPD95.75	50 / 11.8 / 4 = 1.06 μL XbaI/EcoRI pPD95.75
   Fragment 2	46.76 / 120 / 4 = 0.10 μL cel-osm-9p	89.23 / 95.4 / 4 = 0.23 μL cel-tax-4p
   Fragment 3	79.79 / 117 / 4 = 0.17 μL bma-osm-9	44.43 / 95.8 / 4 = 0.12 μL bma-tax-4
   NEBuilder MasterMix	2.5 μL	2.5 μL
   H₂O	1.17 μL	1.09 μL
   Total	5 μL	5 μL

5. Set the thermocycler to incubate at 50°C for 15 minutes for 2–3 fragments, or 60 minutes for 4–6 fragments. Store the assembly at -20°C, or on ice for immediate transformation.

Transformation

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Before you start: heat the water bath to 42°C, remove one LB/Amp/IPTG/X-gal plate (or other selection plate) per assembly reaction from the 4°C incubator, move SOC to room temperature, and move NEB 5-alpha chemically competent cells from the -80°C freezer to ice.

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1. Aliquot 50 μL of the cells to a chilled 1.5 mL tube. Return the competent cells to the -80°C freezer.