Bio-Rad Helios® Gene Gun System User Manual
Page 23
Table 2. Microcarriers and DNA Required for Various Microcarrier
Loading Quantities (MLQ) and DNA Loading Ratios (DLR)
1
Calculated Particle
Materials Required for Selected
Delivery Conditions
MLQ’s and DLR’s
MLQ
DLR
Final
(µg/
(µg/
Gold
DNA
volume
Tubing
(mg/shot)
mg gold)
shot)
(mg)
(µg)
(ml)
2
(total in)
3
0.5
2
1
50 100
6.0
50
0.125
8
1
12.5
100
6.0
50
0.25
4
1
25 100
6.0
50
0.75
1.33
1
75
100
6.0
50
1.0
1
1
100
100
6.0
50
0.5
0.002
0.001
50
0.1
6.0
50
0.5
0.02
0.01
50 1
6.0
50
0.5
0.2
0.1
50
10
6.0
50
0.5
10
5
20 200
2.4
20
1 For most applications with mammalian cells, in initial experiments, use an MLQ of 0.5 and a DLR of 2.
2 Based on loading 1 ml of the DNA-coated microcarriers suspended in ethanol in 8.5 inches (22 cm) of Gold-Coat tubing.
3 Various lengths of tubing may be prepared. Adjust amounts of gold, DNA volume of ethanol in proportion to the change from the
length of tubing listed above for each desired MLQ and DLR. Approximately 25 inches of tubing can be prepared in the Tubing
Prep Station at one time; 50 inches 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 sur-
face of the Gold-Coat 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 pro-
cess. At higher discharge pressures, preparing cartridges 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 recommenda-
tions 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 microfuge tubes
0.05 M spermidine
1 M CaCl
2
200 µl and 500 µl pipettors and tips
5 ml, 10 ml pipettes and pipette-aid
Purified plasmid DNA resuspended in distilled water or 10 mM Tris (pH 8.0), 1 mM EDTA
Ultrasonic cleaner (e.g., Fisher FS3, Branson 1210)
Analytical balance capable of weighing microgram quantities
Microfuge
19