User:David Benjamin Nyer/Notebook/PcTF breast cancer/2016/06/22

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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.