Bio-Rad Helios® Gene Gun System - Manual (Page 22)

5.2 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 Gold-Coat
tubing, it is necessary to calculate the amount of DNA and gold required for each transfor-
mation. 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 agglom-
eration of the gold particles, 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. For delivering 0.5 mg of microcarriers per target (MLQ=0.5), resuspend the DNA/micro-
carrier 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 a MLQ of 0.5 mg/cartridge, a DLR of 2 µg DNA/mg gold results in loading 1 µg of
DNA/cartridge and in delivery of 1 µg of DNA per target. Preparation of two lengths of
Gold-Coat 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 5.2, 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 sev-
eral mammalian targets, refer to Section 7.
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