Summary
Received four polymers from Kaushal lab for transfecting stubborn cell lines. Going to try them out at varying ratios of polymer:DNA with the MCF10A cell lines.
Polymers
Four polymers received:
- AGC18 (1:5): apramycin monomer with glycerol diglycidyl ether cross-linkers, 18 carbons in the lipid conjugate, lipid is conjugated to the polymer backbone at a 1:5 ratio.
- NGC18 (1:5): neomycin monomer with glycerol diglycidyl ether cross-linkers, 18 carbons in the lipid conjugate, lipid is conjugated to the polymer backbone at a 1:5 ratio.
- NR: neomycin monomer with resorcinol diglycidyl ether cross-linkers.
- PGC18 (1:5): paromomycin monomer with glycerol diglycidyl ether cross-linkers, 18 carbons in the lipid conjugate, lipid is conjugated to the polymer backbone at a 1:5 ratio.
Procedure
All polymers were received as 30 mg aliquots, solid, in glass test tubes. Samples are stable at -20 C for at least 6 months in this form.
Prepare polymer stock solutions
Need to make a 2 mg/mL stock solution for each polymer. Do so by weighing out 2 mg in a tared 1.5 mL centrifuge tube, then adding 1 mL of 1x PBS. Polymer NR is slightly hydrophobic, may require resuspension by incubation at room temperature on a rotator for 20-30 minutes.
Prepare DNA:polymer conjugates
Try using 10:1, 25:1 and 50:1 ratios of polymer:DNA for the screen. See the chart below:
| Sample ID
|
Polymer type
|
Ratio
|
Polymer amt (µL)
|
DNA type
|
DNA amt (µg)
|
DNA amt (µL)
|
PBS amt (µL)
|
Total volume (µL)
|
| 1 |
AGC18 (1:5) |
1:10 |
10 |
KAH126-MV2 |
2 |
4 |
86 |
100
|
| 2 |
AGC18 (1:5) |
1:25 |
25 |
KAH126-MV2 |
2 |
4 |
71 |
100
|
| 3 |
AGC18 (1:5) |
1:50 |
50 |
KAH126-MV2 |
2 |
4 |
46 |
100
|
| 4 |
NGC18 (1:5) |
1:10 |
10 |
KAH126-MV2 |
2 |
4 |
86 |
100
|
| 5 |
NGC18 (1:5) |
1:25 |
25 |
KAH126-MV2 |
2 |
4 |
71 |
100
|
| 6 |
NGC18 (1:5) |
1:50 |
50 |
KAH126-MV2 |
2 |
4 |
46 |
100
|
| 7 |
NR |
1:10 |
10 |
KAH126-MV2 |
2 |
4 |
86 |
100
|
| 8 |
NR |
1:25 |
25 |
KAH126-MV2 |
2 |
4 |
71 |
100
|
| 9 |
NR |
1:50 |
50 |
KAH126-MV2 |
2 |
4 |
46 |
100
|
| 10 |
PGC18 (1:5) |
1:10 |
10 |
KAH126-MV2 |
2 |
4 |
86 |
100
|
| 11 |
PGC18 (1:5) |
1:25 |
25 |
KAH126-MV2 |
2 |
4 |
71 |
100
|
| 12 |
PGC18 (1:5) |
1:50 |
50 |
KAH126-MV2 |
2 |
4 |
46 |
100
|
Combine DNA and polymer in each of 12 labeled tubes, then allow to incubate at room temperature for 30 minutes.
Transfect Cells
Add polymer:DNA conjugate to respective wells of cells in a 6-well plate. Mix by rocking the plate, then centrifuge at 100 g for 5 minutes. Return to CO2 incubator.
|
PcTF breast cancer
