Half-Inch Cartridges Using the Tube Cutter
Alex Zorychta (Talk | contribs)
(New page: ==Overview== Detailed below is the procedure to prepare 0.5" cartridges using the tube cutter. Adapted from the [http://www.bio-rad.com/LifeScience/pdf/Bulletin_9541.pdf Instruction Manua...)
Next diff →
Detailed below is the procedure to prepare 0.5" cartridges using the tube cutter. Adapted from the Instruction Manual.
This procedure requires only a few minutes and should be performed as soon after loading the DNA/microparticle suspension into the tubing as possible. It is important to store the coated tubes in a desiccated environment. Tubes stored at 4 °C are stable for at least 8 months.
- Tubing Cutter
- Razor blade
- Cartridge storage vial
- Desiccant pellets
- To be Supplied by User
- [Gold-Coat tubing coated with microcarriers]
- Marking pen
- Analytical Balance
Time considerations: preparation of the DNA/gold suspension requires approximately 30 min. Several samples may be prepared simultaneously without a significant increase in time.
- Prepare a stock solution of 20 mg/ml PVP in ethanol in a small screw-cap container. Dilute this solution with ethanol to prepare PVP solutions at the desired concentration (generally 0.01–0.1 mg/ml); prepare 3.5 ml of the dilute solution for each 30" length of Gold-Coat tubing, (25” to be coated) in the Tubing Prep Station. Keep these solutions tightly capped when not in use. Prepare solution daily.
- In a 1.5 ml microfuge tube, weigh out gold microcarriers. (Refer to Procedure 1 for a detailed description on determining MLQ. Refer to Table 2 for suggestions on the relative amounts of gold and microcarriers required and on the length of tubing produced.)
- To the measured gold, add 100 µl of 0.05 M spermidine. (However, if the volume of plasmid to be added in step 5 is greater than 100 µl, refer to the discussion above for Procedure 2: Determining the DNA Loading Rate, and add the appropriate volume of spermidine.
- Vortex the gold and spermidine mixture for a few seconds, then sonicate for 3–5 seconds using an ultrasonic cleaner to break up gold clumps.
- To the gold and spermidine mixture, add the required volume of plasmid to achieve the desired DLR. (Refer to Procedure 2 for a detailed description on determining DLR. Refer to Table 2 for suggestions on the relative amounts of gold and microcarriers required and on the length of tubing produced.) For co-transfection of multiple plasmids, add each of the plasmids at this step. DNA does not associate with the microcarriers prior to addition of CaCl2.
- Mix DNA, spermidine and gold by vortexing ~5 sec.
- While vortexing the mixture at moderate rate on a variable speed vortexer, add 100 µl of 1 M CaCl2 dropwise to the mixture. The volume added should equal that of the spermidine in Step 3.
- Allow the mixture to precipitate at room temperature for 10 min.
- Most of the gold will now be in the pellet, but some may be on the sides of the tube. The supernatant should be relatively clear. Spin the microcarrier solution in a microfuge ~15 sec to pellet the gold. Remove the supernatant and discard.
- Resuspend the pellet in the remaining supernatant by vortexing briefly. Wash the pellet three times with 1 ml of fresh 100% ethanol each time; spin ~5 sec in a microfuge between each wash. Discard the supernatants.
- After the final ethanol wash, resuspend the pellet in 200 µl of the ethanol solution containing the appropriate concentration of PVP prepared in step 1. Transfer this suspension to a 15 ml disposable polypropylene centrifuge tube with a screw cap. Rinse the microfuge tube once with 200 µl with the same ethanol/PVP solution and add to the centrifuge tube. Add the necessary volume of the ethanol/PVP solution to the centrifuge tube to bring the DNA/microcarrier solution to the desired MLQ.
- The suspension is now ready for tube preparation. Alternatively, the DNA/microcarrier suspensions can be stored for up to 2 months at -20 °C. Prior to freezing, tighten the cap securely and put Parafilm® around the cap of the tube. After storage at -20 °C, allow the particle suspension to come to room temperature prior to breaking the Parafilm seal.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- Who has experience with this protocol?
or instead, discuss this protocol.