Molecular Tests

II Dnases degradation

1.Incubate at 37℃ for 1h, after mixing the protein and plasmid(1μg). The mass ratio between protein and plasmid is 0, 2, 4, 6, 8, 10.

2.Put 1μl DNaseI (TaKaLa) and 10Xbuffer in every EP tube for 30mins.

3.1% agarose gel electrophoresis to test DNA degradation.

IV RT-PCR

1.Prepare the following reaction mixture

dNTP Mixture (10mM each) 1μl

Oligo dT Primer (2.5uM) 1μl

Template RNA 8μl

2.Place tubes in the Thermal Cycler and set the parameters according to the following conditions for denaturation and annealing.

65℃ for 5min.

4℃

3.Prepare the reagent mixture for use in step 2 by combining the reagents in the proportions shown as below.

Reaction mixture used for denaturation and annealing from A-2 10μl

5X PrimeScript Buffer 4μl

RNase Inhibitor (40U/μl) 0.5μl

PrimeScript RTase (for 2 step) 0.5μl

RNase Free dH2O 5μl

4.Place the tubes in a Thermal Cycler and perform reverse transcription using the following program

42℃ 15-30min

95℃ 5min

4℃

5.Prepare reaction miture by combining the following reagents.

10XPCR Buffer II 5μl

dNTP Mixture(10mM each) 2μl

Upstream Primer 0.5μl

Downstream Primer 0.5μl

TaKaRa Ex Taq HS 0.5μl

Reverse transcriptant from step4 5μl

H20 up to 50μl

6.After mixing reagents, put all tubes in the thermal cycler, and start PCR under the following program.

94℃ 30s

57℃ 30s

72℃ 30s

For 30 cycles

7.After PCR, load 2ul to a 1% agarose gel to have a quick run checking the production of desired fragment.

Cellular Test

• Molecular Tests
• Cellular Test