How to make Tol2 mRNA

Find the Tol2 plasmid from the Wilson lab freezer. It is #1151 and is labelled Tol2.

The concentration (measured on Qubit) is ~ 690 ng/µL (as of the Eppendorf in 2022).

Thaw it.

We linearise the plasmid using the restriction enzyme NotI HiFi.

On ice, prepare 6 reactions in 0.5 mL Eppendorfs.

Each reaction is (pipet in this order):

• Tol2 plasmid 2.9 µL
• NotI HiFi 2 µL
• CutSmart buffer 10× 10 µL
• nuclease-free H2O 91.1 µL Total = 100 µL

(Add the enzyme last).

Place the Eppendorfs in the 37˚C water bath.

Incubate for at least 1 hour. Digesting for more than 1 hour may be beneficial (cf. https://international.neb.com/tools-and-resources/usage-guidelines/restriction-endonucleases-survival-in-a-reaction).

Clean (remove the enzyme etc.) the reaction using the QIAquick PCR Purification Kit. Similar column-based kits are probably OK.

Follow the protocol in the box or online.

Use a single column.

For the first step (adding 5 volumes of PB buffer): transfer each 3 reactions in a 2 mL Eppendorf to obtain two 2 mL Eppendorfs, each containing 300 µL of reaction.

Add 1500 µL (5 volumes) of PB buffer. Vortex and spin down (with the benchtop centrifuge).

Add ~ 700 µL on the column and spin down following the kit's instructions (big centrifuge). Discard flow-through. Repeat until you got all 600 µL of reaction (the two 2 mL Eppendorfs) through the column.

At the last step of the kit's protocol, elute in 30 µL nuclease-free H2O.

Measure the concentration on the Qubit (dsDNA BR or HS kit). We expect the concentration to be 200–400 ng/µL *.

Next step is to turn the linearised Tol2 plasmid (DNA) into mRNA. Find the SP6 mMESSAGE mMACHINE kit.

! Make sure you are using the SP6 version.

In my experience, each reaction generates 3,000–4,000 ng mRNA. Therefore, to get ~ 12 µL of > 1,000 ng/µL, you will need at least 3 reactions. This is assuming a good output and losing nothing on the column during clean-up, so I try to do 5–6 reactions for critical injections where I need high integration rate directly in F0 injected embryos.

Prepare each reaction on ice in a small 0.5 mL Eppendorf.

Each reaction is:

• 2× NTP/CAP 10 µL
• 10× reaction buffer 2 µL
• linearised Tol2 plasmid ***
• enzyme mix 2 µL
• nuclease-free H2O to 20 µL

*** Calculate from the concentration found in the previous step so that you use 1 µg.

e.g. I found the concentration to be 343 ng/µL, so I use 2.91 µL here.

Place the Eppendorfs in the 37˚C water bath.

Incubate for 2 hours.

There is no benefit in incubating for more than 2 hours (cf. reaction time course: https://www.thermofisher.com/order/catalog/product/AM1340#:~:text=mMESSAGE%20mMACHINE%E2%84%A2%20kits%20are,structure%20at%20the%205'%20end.). Should be OK if reaction lasts a bit longer if for whatever reason you cannot stop it in time.

Add 1 µL TURBO DNase in each reaction (included in the kit). This is removes the DNA, i.e. the linearised plasmid.

Incubate in the 37˚C water bath for 15 min.

Clean-up the mRNA using the ZYMO RNA Clean & Concentrator kit.

Follow the kit's instructions. Use a single column.

At the end, elute in 13 µL nuclease-free H2O. Add the 13 µL directly on the column's filter and wait for a few minutes at room temperature.

Heating the nuclease-free H2O to 70˚C prior to adding it to the water may also help with retrieving as much as possible mRNA from the column, but I have never tried.

(You can always dilute later if you find that the Tol2 mRNA is highly concentrated, so best to elute in a small volume.)

Measure the concentration on Qubit (BR or HS RNA kit). Qubit can measure low concentrations, so no need to waste precious Tol2 mRNA. You can e.g. dilute 10×:

• 0.4 µL Tol2 mRNA
• 3.6 µL nuclease-free H2O and measure that concentration. Just remember to multiply the concentration you find by 10.

You are expecting (hoping) > 1,000 ng/µL. For reference, if each of 6 reactions produced 3000 ng and nothing is lost on the column, you would expect ~ 1384 ng/µL (6 × 3000 ng, eluted in 13 µL).

Aliquot the ~ 13 µL in 3–4 small 0.5 mL Eppendorfs and store in the –80˚C freezer.