Lab meeting 13.03.31

1st Plasmid preparation of K562, Hela, IM9 -Midi Kit Quiagen

2nd 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)

K1

K2

K3

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/µlK3: 21 ng/µl H3: 20 ng/µl I3: 14 ng/µl

A260/280 : 2.012A260/230: 2.011

pcDNA3.1 + cDNA U2AF1: 5989 bp K1 K2 K3 H1 H2 H3 I1 I2 I3 Ladder

K562 Hela IM9

5000bp

1000bp

Result of plasmid preparation

Plasmid form Cell line Concentration (ng/µl)

Abs260/Abs280 Abs260/Abs230 Volume(µl)

Open circular & supercoil form[pcDNA3.1+

cDNA U2AF1]

K562 2098 2.2 2.5 20

Hela 2414 2.2 2.5 20

IM9 1681 2.14 2.44 20

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

pcDNA3.1 digest with ScaI- blunt end

• Dilute [pcDNA3.1+cDNA U2AF1] up to 20µl (1µg/µl)

Linearize pcDNA3.1+cDNA U2AF1 by ScaI

ScaI 2µl

10X NEB buffer (No.3) 10µl

BSA 3µl

pcDNA3.1+cDNA (1µg/µl) 20µl

D.W 15µl

Total 50µl

• Incubation time: O/N• Incubation temp: 37 °C

Plasmid form Cell line Concentration (ng/µl)

Abs260/Abs280 Abs260/Abs230 Volume(µl)

K562 240 2.0 2.4 15

Hela 200 2.0 2.4 15

IM9 200 2.0 2.4 15

Result of plasmid linearization by ScaI

Ladder K562 Hela IM9 • pcDNA3.1 + cDNA U2AF1: 5989 bp