Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/06/03: Difference between revisions

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==Procedure==
==Procedure==


# Measure cell density, spin down and resuspend in 5 mL PBS (15 mL conical), spin down again and resuspend in at least 0.8 mL Resuspension Buffer R (1.5 mL microcentrifuge tube) to bring total cell density to 2.0E7 cells/mL.
# Measure cell density, spin down and resuspend in 5 mL PBS (15 mL conical), spin down again and resuspend in at least 800 µL Resuspension Buffer R (1.5 mL microcentrifuge tube) to bring total cell density to 2.0E7 cells/mL.
# Set up two sample tubes, labeled 'GFP (+)' and 'sample'. To the GFP tube add 30 µL of pMaxGFP at 2.0 µg/µL. To the sample tube add 50 µL of KAH126-MV2 at 2.0 µg/µL.
# Set up two sample tubes, labeled 'GFP (+)' and 'sample'. To the GFP tube add 30 µL of pMaxGFP at 2.0 µg/µL. To the sample tube add 50 µL of KAH126-MV2 at 2.0 µg/µL.
# Aliquot 300 µL of cells to the GFP tube and 500 µL of cells to the sample tube. Mix by gentle pipetting up and down each time.
# Aliquot 300 µL of cells to the GFP tube and 500 µL of cells to the sample tube. Mix by gentle pipetting up and down each time.

Revision as of 11:09, 3 June 2015

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Overview

On Monday I started a 20 mL culture of K562 in antibiotic-free medium. Today I'll be checking cell density and planning out the transfection setup.

Cell Density

Measured at 300,000 cells/mL by hemacytometer.

3.0E5 cells/mL * 20 mL = 6.0E6 cells total

Experimental Design

The plan is to collect 1 control (GFP) and 2 samples (KAH126-MV2) for each day of collection, and to collect on days 2 and 3. This makes 6 samples total. I'll be using a 6-well plate for this experiment and transfecting using the 100 µL tips.

Cells will be resuspended in buffer at 2.0 x 107 cells/mL for transfection. Multiplied by 8 transfections (2 buffer), that's 2.0E7 cells/mL * 0.8 mL = 1.6E7 cells needed.

Number of cells per well: 0.4-1 x 106

Initial cell density after transfection and transferring to wells (in 2 mL plating medium) will be 2.0E7 cells/mL * 0.1 mL / 2.0 mL = 1.0E6 cells/mL

Amount of DNA per sample is 5-30 µg, not to exceed 10% of total volume of cells (100 µL * 0.1 = 10 µL). Both pMaxGFP and KAH126-MV2 are at concentrations of 2.0 µg/µL, so 10 µL per sample would be 20 µg of DNA per sample.

Procedure

  1. Measure cell density, spin down and resuspend in 5 mL PBS (15 mL conical), spin down again and resuspend in at least 800 µL Resuspension Buffer R (1.5 mL microcentrifuge tube) to bring total cell density to 2.0E7 cells/mL.
  2. Set up two sample tubes, labeled 'GFP (+)' and 'sample'. To the GFP tube add 30 µL of pMaxGFP at 2.0 µg/µL. To the sample tube add 50 µL of KAH126-MV2 at 2.0 µg/µL.
  3. Aliquot 300 µL of cells to the GFP tube and 500 µL of cells to the sample tube. Mix by gentle pipetting up and down each time.
  4. Proceed with Neon Transfection following the instructions on the back of the quick reference card. There should be an extra 50 µL per GFP control well and an extra 25 µL per KAH126-MV2 sample well.
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