BIO RAD Helios Gene Gun System Instruction Manual page 25

Table 2. Microcarriers and DNA required for various microcarrier loading quantities (MLQ) and DNA loading ratios (DLR).*
Calculated Particle Delivery Conditions
MLQ, DLR,
mg/shot µg/mg gold
0.5 2
0.125 8
0.25 4
0.75 1.33
1.0 1
0.5 0.002
0.5 0.02
0.5 0.2
0.5 10
* For most applications with mammalian cells, in initial experiments, use an MLQ of 0.5 and a DLR of 2.
** Based on loading 1 ml of the DNA-coated microcarriers suspended in ethanol in 8.5" (22 cm) of GoldCoat Tubing.
*** Various lengths of tubing may be prepared. Adjust amounts of gold and DNA volume of ethanol in proportion to the change from the length of
tubing listed above for each desired MLQ and DLR. Approximately 25" of tubing can be prepared in the Tubing Prep Station at one time; 50"
of tubing will usually yield 80–90 cartridges.
Precipitation of DNA onto Microcarriers
It is important to use an unopened bottle of 100% ethanol each day this step is performed. Opened bottles
of ethanol absorb water and the presence of water in the tubing while drying will lead to streaking, clumping,
and uneven coating of the microcarriers over the inner surface of the GoldCoat Tubing, resulting in poor or
unusable cartridges. All ethanol solutions should be opened only briefly when in use and kept tightly capped
when not in use.
Polyvinylpyrrolidone (PVP) serves as an adhesive during the cartridge preparation process. At higher
discharge pressures, cartridges prepared with PVP can increase the total number of particles delivered.
The optimum amount of PVP to be used must be determined empirically. Typical PVP concentrations range
from 0.01 to 0.1 mg/ml. For recommendations on the amount of PVP to use in initial experiments, refer
to Section 7.
Materials
Provided
■
− Gold microcarriers
− Polyvinylpyrrolidone (PVP), 360,000 MW
To be supplied by user
■
− Fresh 100% ethanol
− 15 ml disposable polypropylene centrifuge tubes
− 1.5 ml microcentrifuge tubes
− 0.05 M spermidine
− 1 M CaCl
2
− 200 and 500 µl pipettors and tips
− 5 ml, 10 ml pipets and pipet aid
DLR,
Gold, mg
µg/shot
1 50
1 12.5
1 25
1 75
1 100
0.001 50
0.01 50
0.1 50
5 20
Section 5 Operation of the Helios Gene Gun System
Materials Required for Selected MLQs and DLRs
Final volume,
DNA, µg
100
100
100
100
100
0.1
1
10
200
Tubing, total in***
ml**
6.0 50
6.0 50
6.0 50
6.0 50
6.0 50
6.0 50
6.0 50
6.0 50
2.4 20
Helios Gene Gun System | 21