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

PcTF breast cancer

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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.