Template:Heidelberg/notebook/pc/week35

week number 35

▼2015-08-25 RNA extraction

Because the functionality of the ribozyme couldn’t be shown on protein level, the next step is to show functionality on RNA level. Therefore RT-PCR was performed with positive control samples and SH09 samples. Old means that the samples were also used for the Western Blot described above.

RNA extraction

1. Grow yeast cells in 10ml of desired medium to mid-exponential phase (OD600=1.0).

2. Transfer culture to 50-ml centrifuge tube and centrifuge cells 3min at 1500g, 4°C

3. Discard supernatant, resuspend pellet in 1ml ice-cold water. Transfer to clean 1.5-ml microcentrifuge tube. Microcentrifuge 10 sec at 4°C, and remove supernatant.

4. Resuspend pellet in 300µl RNA buffer: 0.5M NaCl, 200mM Tris Cl, pH 7.5, 10mM EDTA

Disrupt the cells

5. Add a volume of chilled acid-washed glass beads equivalent to 200ml water

6. Add 300µl of 25:24:1 phenol/chloroform/isoamyl alcohol equilibrated with RNA buffer.

7. Close the cap, then invert and shake up and dwon to ensure that the beads are suspended. Vortex vigorously for 2min at highest speed.

8. Microcentrifuge 1min at room temperature. Transfer aqueous layer to a clean microcentrifuge tube.

9. Add an equal volume of 25:24:1 phenol/chloroform/isoamyl alcohol. Vortex vigorously 10 sec.

10. Repeat step 8.

Precipitate the RNA

0.1 volume of 3M NaAC

2.5 volume of ethanol

Incubate at least 2 hours at -20°C, centrifuge for 30min at 4°C full speed. Discard supernatant and resuspend pellet in 20µl water.

DNA digest, Precipitate the RNA as described above.

▼2015-08-26 RT-PCR

4µl RNA

1µl dNTPmix (10mM each)

1µl primer (2µM)

7µl water

 

65°C, 5min, on ice, spin down

4µl 5xFirst buffer

1µl 0.1M DTT

1µl RNase inhinitor

1µl SuperScript III RT or for –RT control 1µl water

 

55°C, 1h for gene specific primer: xxJD33RTmycxx, xxJD34RTgfpxx

50°C oligo dT

70°C, 15min

150mM NaOH, 37°C, 15min

150mM HCl

DNA precipitation with EtOH and NaOAc as described above.

▼2015-08-27 PCR with cDNA

With all four samples (pos old, pos new, ribozyme old, ribozyme new) PCRs with following primers were performed:

xxDH076xx and xxJD034xx

xxDH076xx and xxJD033xx

SH021 and JD033xx

Program:

94°C 4min

94°C 30 sec

60°C 30sec

72°C 45 sec

72°C 10min

4°C for ever

 

Samples were sent for sequencing.

▼2015-08-28 Sequence information

Sequence of the ribozyme can be confirmed. Sequencing doesn’t show any ribozyme product: GFP-myc in ribozyme samples.