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)
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
• 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