BIO RAD Helios Gene Gun System, Инструкция по эксплуатации

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Section 5 Operation of the Helios Gene Gun System
20 | Helios Gene Gun System
After the Bombardment
1. Remove cartridge holder from gene gun.
2. Remove cartridges from cartridge holder.
3. Turn off the helium pressure to the system.
4. Turn the regulator value counterclockwise to depressurize the system.
5. Disconnect the helium hose and gene gun.
Preparation of System Components Prior to Bombardment
Calculating the Amounts of Gold and Plasmid Required
Prior to precipitating DNA onto the gold particles and loading them into the GoldCoat Tubing, it is necessary
to calculate the amount of DNA and gold required for each transformation. Points to consider in making
these calculations are presented below. The amount of DNA loaded per mg of microcarriers is referred to as
the DNA loading ratio (DLR). Typical DLRs range between 1 and 5 µg DNA/mg gold. Adding more
DNA tends to cause the gold particles to agglomerate, probably as a result of DNA binding to more than
one particle. The amount of microcarriers delivered per target is referred to as the microcarrier loading
quantity (MLQ). Typical MLQs range from 0.25 to 0.5 mg/cartridge for in vivo
delivery to epidermal cells,
but may be slightly lower for in vitro delivery to mammalian cells. Refer to Table 2 for representative
starting amounts of microcarriers and plasmid to use for different MLQs and DLRs. Refer to Section 7 for
suggestions on parameter optimization and starting conditions for using the Helios Gene Gun to deliver
DNA to mammalian cells.
Procedure 1: Determining the Microcarrier Loading Quantity (MLQ)
1. For most systems, delivering 0.5 mg of gold per target is a good starting point.
2. A 1 ml suspension will fill an 8.5" length of tubing; one cartridge is 0.5" long. Each 30" length of tubing
can be filled with approximately 25" (3.0 ml) of DNA/gold suspension. (There will be a void space at
each end.)
3. To deliver 0.5 mg of microcarriers per target (MLQ = 0.5), resuspend the DNA/microcarrier sample at
8.5 mg of gold/ml ethanol. A 25" length of tubing will require 25 mg of gold resuspended in a volume of
3 ml of ethanol.
Procedure 2: Determining the DNA Loading Ratio (DLR)
1. For many applications, delivery of 1 µg of plasmid per target is a good starting point.
2. At an MLQ of 0.5 mg/cartridge, a DLR of 2 µg DNA/mg gold results in loading 1 µg of DNA/cartridge
and delivering 1 µg of DNA per target. Preparing two lengths of GoldCoat Tubing requires
100 µg of DNA and 50 mg of gold. The concentration of DNA should be approximately 1 µg/µl and the
volume of DNA should not exceed the volume of spermidine in Section 5, Precipitation of DNA onto
Microcarriers, step 3. If the DNA is too dilute, concentrate it by ethanol precipitation. If a high DLR is
desired, increase the volume of spermidine and CaCl
2
so that equal volumes of each component are
added (spermidine, DNA, and CaCl
2
) up to a total volume of 1,200 µl.
3. For a detailed description on determining which MLQs and DLRs will work best for several mammalian
targets, refer to Section 7.