1. Select transformants on LB plates containing 100 μg/ml ampicillin  Successful colonies K562: 27 colonies Hela: 11 colonies IM9: 25 colonies Lab Meeting 13.03.18

2. Sequencing  pcDNA3.1 + cDNA was sequenced in both forward and reverse direction  confirm Forward Reverse  Successful colonies K562: 2/27 (No.1, 4) Hela: 3/11 (No.2, 3, 11) IM9: 4/25 (No.1, 2, 5, 13) No mutation Full length sequence was cloned Proper orientation

3. Analyze transformants for the presence of insert 1 4 2 3 11 1 2 5 13 K562HelaIM9 5000bp 500bp pcDNA3.1 : 5428 bp Cloned cDNA U2AF1 : 552 bp

4. Next step

5. Determining antibiotic sensitivities  Protocol: 1. Plate or split a confluent plate so the cells will be approximately 25% confluent. Prepare a set of 6–7 plates. Add the following concentrations of antibiotic to each plate: – For Geneticin® selection, test 0, 50, 125, 250, 500, 750, and 1000 μg/ml Geneticin®. 2. Replenish the selective media every 3–4 days, and observe the percentage of surviving cells. 3. Count the number of viable cells at regular intervals to determine the appropriate concentration of antibiotic that prevents growth within 1–3 weeks after addition of the antibiotic.  Once the appropriate Geneticin concentration to use for selection in host cell line was detected  possible to generate a stable cell line expressing gene of interest. Source: http://tools.invitrogen.com/content/sfs/manuals/pcdna3_1_man.pdf  Determine the minimum concentration of Neomycin required to kill untransfected host cell line (K562, Hela, IM9)  Test a range of concentrations to determine the minimum concentration necessary for cell line.

6. Set up protocol for determine Neomycin sensitivities of Hela 50 μg/ml 125 μg/ ml 125 μg/ ml 250 μg/ ml 500 μg/ ml 750 μg/ml 1000 μg/m l Control  split a confluent plate so the cells will be approximately 25% confluent  Neomycin 10mg/ml  A1-3 : 500 μg/ml cells + 0 μg/ml Neo + 500 μg/ml RPMI 10% FBS P.S  A4-6 : 500 μg/ml cells + 5 μg/ml Neo + 495 μg/ml RPMI 10% FBS P.S  B1-3 : 500 μg/ml cells + 12,5 μg/ml Neo + 487,5 μg/ml RPMI 10% FBS P.S  B4-6 : 500 μg/ml cells + 25 μg/ml Neo + 475 μg/ml RPMI 10% FBS P.S  C1-3 : 500 μg/ml cells + 50 μg/ml Neo + 450 μg/ml RPMI 10% FBS P.S  C4-6 : 500 μg/ml cells + 75 μg/ml Neo + 425 μg/ml RPMI 10% FBS P.S  D4-6 : 500 μg/ml cells + 100 μg/ml Neo + 450 μg/ml RPMI 10% FBS P.S  Total volume of Hela cells + selective medium : 1 mL  Replenish the selective media every 3–4 days, and observe the percentage of surviving cells.  Count the number of viable cells at regular intervals to determine the appropriate concentration of antibiotic that prevents growth within 1–3 weeks after addition of the antibiotic.

7. 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

8. Electroporation

9. Selection of stable integrant  Once the appropriate Geneticin concentration to use for selection in host cell line was detected  possible to generate a stable cell line expressing gene of interest. 1. 24 hours after transfection, wash the cells and add fresh medium to the cells. 2. 48 hours after transfection, split the cells into fresh medium containing Neomycin at the predetermined concentration required for cell line. Split the cells such that they are no more than 25% confluent. 3. Feed the cells with selective medium every 3–4 days until Geneticin-resistant foci can be identified. 4. Pick and expand colonies in 96- or 48-well plates.