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

Sequencing pcDNA3.1 + cDNA was sequenced in both forward and reverse direction

confirm

Forward Reverse

Successful coloniesK562: 2/27 (No.1, 4)Hela: 3/11 (No.2, 3, 11)IM9: 4/25 (No.1, 2, 5, 13)

No mutationFull length sequence was clonedProper orientation

Analyze transformants for the presence of insert

1 4 2 3 11 1 2 5 13

K562 Hela IM9

5000bp

500bp

• pcDNA3.1 : 5428 bp

• Cloned cDNA U2AF1 : 552 bp

Next step

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.

Set up protocol for determine Neomycin sensitivities of Hela

50 μg/ml

50 μg/ml

50 μg/ml

125 μg/ml

125 μg/ml

125μg/ml

250 μg/ml

250 μg/ml

250 μg/ml

500 μg/ml

500 μg/ml

500 μg/ml

750 μg/ml

750 μg/ml

750 μg/ml

1000 μg/ml

1000 μg/ml

1000 μg/ml

Control Control 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.

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

Electroporation

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.