1. Lab meeting 13.03.31  1 st Plasmid preparation of K562, Hela, IM9 -Midi Kit Quiagen  2 nd Prepare control sample within the plasmid preparation process Each sample of control was prepared as following: K1: cleared lysate containing supercoiled and open circular plasmid DNA & degraded RNA (240µl) K2: second wash fraction, which ensure that the resin is completely cleared of RNA & other contaminants, leaving only pure plasmid DNA on the column (400µl) K3: the eluate containing pure plasmid DNA with no other contaminating nucleic acids (100µl) Prepare same control sample for Hela (H1, H2, H3) and IM9 (I1, I2, I3)

2. K1K2K3

3. Result of control Concentration: K1: 225 ng/µl H1: 178 ng/µl I1: 200 ng/µl K2: 2.7 ng/µl H2: 1.1 ng/µl I2: 3.3 ng/µl K3: 21 ng/µl H3: 20 ng/µl I3: 14 ng/µl A260/280 : 2.012 A260/230: 2.011 pcDNA3.1 + cDNA U2AF1: 5989 bp K1 K2 K3 H1 H2 H3 I1 I2 I3 Ladder K562HelaIM9 5000bp 1000bp

4. Result of plasmid preparation Plasmid formCell lineConcentration (ng/µl) Abs260/Abs280Abs260/Abs230Volume (µl) Open circular & supercoil form [pcDNA3.1+ cDNA U2AF1] K56220982.22.520 Hela24142.22.520 IM916812.142.4420

5. To create stable cell line  Linearize the pcDNA™3.1(+) vector Linearize the pcDNA3.1 + cDNA U2AF1 Source: http://tools.invitrogen.com/content/sfs/manuals/pcdna3_1_man.pdf

6. pcDNA3.1 digest with ScaI- blunt end

7. Dilute [pcDNA3.1+cDNA U2AF1] up to 20µl (1µg/µl) Linearize pcDNA3.1+cDNA U2AF1 by ScaI ScaI2µl 10X NEB buffer (No.3)10µl BSA3µl pcDNA3.1+cDNA (1µg/µl)20µl D.W15µl Total 50µl Incubation time: O/N Incubation temp: 37 °C

8. Plasmid formCell lineConcentration (ng/µl) Abs260/Abs280Abs260/Abs230Volume (µl) K5622402.02.415 Hela2002.02.415 IM92002.02.415 Result of plasmid linearization by ScaI Ladder K562 Hela IM9 pcDNA3.1 + cDNA U2AF1: 5989 bp