by Andreas Torell
This protocol shows how to make nucleofection agents yourself. Together with cuvettes from Sigma Aldrich these buffer is enough to do all nucleofection workflows yourself assuming you have a nucleofector.
The transfection buffer for nucleofection will be made into three separate bufferts that can be frozen and later on combined to make the working buffer for nucleofection. Note that once the partial solutions have been mixed together (Buffer A, B, and C), it is no longer advisable to freeze the buffer due to the formation of insoluble precipitates.
| Item | Item article number | Distributor | Vendor |
|---|---|---|---|
| Ficoll® 400 |
F8016-5G |
SigmaAldrich | Merck |
| PEG 6000 | A17541.30 | ThermoFisher | ThermoFisher |
| Poloxamer 188 solution 10%, 100 mL | P5556-100ML | SigmaAldrich | Merck |
| UltraPure DNase/RNase-Free Distilled Water | 11538646 | Invitrogen | ThermoFisher |
| Monobasic Sodium Phosphate (NaH2PO4) | S0751-500G | SigmaAldrich | Merck |
| Dibasic Sodium Phosphate (Na2HPO4) | S9390-500G | SigmaAldrich | Merck |
| HEPES | H3375-500G | SigmaAldrich | Merck |
| Potassium Chloride (KCl) | 1.04936 | SigmaAldrich | Merck |
| Magnesium Chloride (MgCl2) | M8266-100G | SigmaAldrich | Merck |
| D-Mannitol | 63560-250G-F | SigmaAldrich | Merck |
| Fetal Bovine Serum | 10100147 | Gibco | ThermoFisher |
In three separate 1.5 mL Eppendorf tubes, using an analytical balance, weigh the dry reagents:
| Item | Mass |
|---|---|
| Ficoll | 100 mg |
| PEG 6000 | 50 mg |
| Poloxamer 188 (10%) | 50 uL |
Prepare a 15 mL Falcon tube with 10 mL of nuclease-free water.
Dissolve the Ficoll and the PEG 6000 with an adequate amount of nuclease free water.
Transfer the contents of both Eppendorf tubes (Ficoll and PEG 6000) to a new sterile 15 mL Falcon tube, add the 50 uL of Poloxamer 188 to this Falcon tube and pipette mix.
Fill up to 10 mL with nuclease-free water and mix the content using a tube-inverter until the solution becomes homogenous.
DO NOT vortex the buffer if you are having trouble with dissolving the reagents, instead heat it up to 37°C and then continue inverting the tube.
Prepare a 15 mL Falcon tube with 8 mL of nuclease-free water.
Using an analytical balance, weigh up 692 mg of NaH2PO4 and 2.68 g of Na2HPO4 and add both reagents to the water-filled Falcon tube.
Vortex the solution until the salts have completely dissolved and then dilute the solution to a final volume of 10 mL with water.
If the salts are difficult to dissolve, heat the solution to around 40°C before vortexing.
Fill up to 10 mL with nuclease-free water and mix the content using a tube-inverter until the solution becomes homogenous.
DO NOT vortex the buffer if you are having trouble with dissolving the reagents, instead heat it up to 37°C and then continue inverting the tube.
In an Eppendorf tube make a 151 mM stock of HEPES.
| Reagent | Amount | Final concentration |
|---|---|---|
| HEPES | 36 mg | 151 mM |
| Nuclease-free water | 1 mL | |
| Total: | 1 mL |
In an Eppendorf tube make a 151 mM stock of Potassium Chloride (KCl).
| Reagent | Amount | Final concentration |
|---|---|---|
| KCl | 37 mg | 496 mM |
| Nuclease-free water | 1 mL | |
| Total: | 1 mL |
In an Eppendorf tube make a 1.5 M stock of Magnesium Chloride (MgCl).
| Reagent | Amount | Final concentration |
|---|---|---|
| MgCl2 | 143 mg | 1.5 M |
| Nuclease-free water | 1 mL | |
| Total: | 1 mL |
In an Eppendorf tube make a 5 M stock of D-Mannitol.
| Reagent | Amount | Final concentration |
|---|---|---|
| D-Mannitol | 911 mg | 5 M |
| Nuclease-free water | 1 mL | |
| Total: | 1 mL |
Fill a 15 mL Falcon tube with 4 mL of nuclease-free water.
Transfer the solutions (HEPES, KCl, MgCl2 and D-Mannitol) to the Falcon tube (one at a time), mixing each solution thoroughly by pipetting before adding the next.
Add 1 mL of FBS to the buffer and fill up the mixture to 10 mL with nuclease-free water.
This will give you a final concentration of the following for Buffer C:
BUFFER C:
| Reagent | Volume | Final concentration |
|---|---|---|
| HEPES (151 mM) | 1 mL | 15.1 mM |
| KCl (496 mM) | 1 mL | 49.6 mM |
| MgCl2 (1.5 M) | 1 mL | 150 mM |
| D-Mannitol (5 M) | 1 mL | 500 mM |
| FBS | 1 mL | |
| Nuclease-free water | 5 mL | |
| Total: | 10 mL |
Sterilise each buffer by passing them through a 0.22 um filter before aliquoting into smaller fractions i.e. Eppendorf tubes.
The partitioned buffers can now be stored indefinitely at -20°C. The solutions are now at a concentration of 10X.
Note: Once the solutions are thawed again, you might get precipitate formation in buffers A and B however, these are reversibly dissolvable through heating and vigorous vortexing. Additionally, buffer C can turn gelatinous after thawing and will subsequently need a slight heating to reach a less viscous homogenic state before use.
Important: Always make sure that each buffer is completely homogenous before using them to make a new batch of transfection buffer. If any precipitates remain after trying to homogenize a fraction, throw it away and thaw a new partition instead.
Making a 2 mL complete transfection buffer from the 3-part stock solutions:
Thaw and homogenise each of the solutions and inspect them closely before use.
Add 1400 uL of nuclease-free water to a sterile 2 mL Eppendorf tube.
Start by transferring 200 uL of Buffer A to the tube and mix thoroughly before proceeding.
Repeat previous step for Buffer B and Buffer C.
The transfection buffer is now complete and can be used instantly or stored at 4°C for approximately 1 month.