Isolation of nucleic acids

2.2.1 Isolation of total RNA

1) Cell lysis: 450 µl of Buffer RLT was added to a maximum of 100 mg of frozen cells and was vortexed vigorously.

2) Homogenization: The lysate was applied to the QIAshredder spin column and was centrfuged at 14000 rpm for 2 min.

3) Ethanol precipitation: The flow-through was carefully transferred to a new tube and was mixed well with 0.5 volumes of 100% ethanol. The sample was then applied to a RNeasy column and centrifuged at 10,000 rpm for 15 sec.

4) Washing: To wash the membran, 350 µl of Buffer RW1 was added and the column was centrifuged for 15 sec at 10,000 rpm.

5) On-column DNase digestion:10 µl of DNase I stock solution was added to 70 µl Buffer RDD and mixed gently by inverting the tube. The mix was directly pipetted onto the RNeasy silica-gel membrane. The column was then incubated at room temperature for 15 min.

6) Washing: The RNeasy column was transferred into a new collection tube and 500 µl Buffer RPE was added. The columne was centrifuged for 15 s at 10,000 rpm. The washing step was repeated once with further 500 µl Buffer RPE. To dry the membrane completely, the column was centrifuged at full speed for 1 min

7) Elution: To elute RNA, the RNeasy column was transferred to a RNase-free 1.5 ml tube and 50 µl RNase-RNase-free water was added.The column was then centrifuged at 10,000 rpm for 1 min. To obtain a higher total RNA concentration, the elution step was repeated by using the first eluate.

2.2.2 Poly A⁺ mRNA isolation from total RNA using Dynabeads^® Oligo(dT)₂₅

2.2.2.1 Principle

Dynabeads Oligo (dT)25 are uniform, superparamagnetic (2,8 µm diameter) with 25 nucleotid-long chains of deoxythymidines covalently linked to their surfaces.

Dynabeads Oligo (dT)25 are designed for rapid isolation of poly A⁺ RNA either from total RNA or directly from crude extracts. The use of Dynabeads Oligo (dT)25 relies on base pairing between the poly A tail of most messenger RNA and the oligo dT sequences. The binding capacity ot the Dynabeads Oligo (dT)25 is 2 µg polyadenylated mRNA per mg Dynabeads.

2.2.2.2 Procedures

1) The Dynabeads Oligo (dT)25 was resuspended by gently flicking the tube and 85 µl of Dynabeads Oligo (dT)25 from the stock suspension was transferred to an RNase-free 1.5 ml microcentrifuge tube.

2) The tube was then placed in magnet stand (Dynal MPC-E-1) for 30 sec.

After the supernatant discarded, the tube was removed from the magnet stand and the Dynabeads was resuspended in 100 µl binding buffer.

3) The tube was placed in magnet stand again to remove the binding buffer.

4) The Dynabeads was resuspended again in 100 µl binding buffer. 25 µg Total RNA was adjusted to 100 µl with DEPC-treated water and was heat at 65°C for 2 min.

5) The total RNA was then mixed to the Dynabeads and thesuspension was incubated on a rotating mixer for 5 min to anneal RNA.

6) The tube was placed in the magnet stand for 30 sec. The supernatant was removed and the Dynabeads was washed twice with 200µl washing buffer.

7) 15 µl of elution buffer was added and the tube was incubate at 65 °C for 2 min. The tube was placed in the magnet stand again and the supernatant containing mRNA was transferred to a new RNase-free tube.

8) To eliminate any ribosomal RNA contamination, the eluted mRNA was reextracted. First, the Dynabeads was washed twice with 200 µl washing Buffer. The beads were then resuspended in 60 µl binding buffer. After incubation on a roller for 5 min at room temperature, the Dynabeads Oligo (dT)25/mRNA was washed twice with 200 µl washing buffer and the mRNA was eluted as above.

2.2.3 Isolation of DNA using QIAquick PCR Purification Kit

1) DNA binding: 5 volumes of Buffer PB was added to 1 volume of the PCR sample and mixed. The QIAquick spin column was loaded with the sample and was centrifuged for 1 min.

2) Washing: 0.75 ml of Buffer PE was added and the column was centrifuged for 1 min.The flow-through was discarded and the column was centrifuged for an additional 1 min.

3) To elute DNA, 50 µl of Buffer EB was added and the column was centrifuged for 1 min. If higher DNA was desired, 30 µl Buffer EB was added and the column was incubated at room temperature for 1 min. The column was then centrifuged as above.

2.2.3 Isolation of DNA from agarose gel using QIAquick Gel Extraction Kit

All centrifuge steps were at 13,000 rpm and at roomtemperature.

1) The DNA fragment was excised from the agarose gel and weighed. 3 volumes of Buffer QG were added to 1 volume of gel. The gel slice was incubated at 50 °C for 10 min. To support dissolving, the tube was mixed by vortexing every 2-3 min.

2) To increase the yield of DNA fragments <500 bp, 1 gel volume of isopropanol was added to the sample and mixed. If the fragment was >500 bp this step was skiped as addition of isopropanol has no effect on yield.

3) The sample was loaded to a QIAquick spin column and the column was centrifuged for 1 min.

4) 0.75 ml of Buffer PE was added and the column was centrifuged for 1 min.

The flow-through was discarded and the column was centrifuged for an additional 1 min.

5) 50 µl of Buffer EB was added and the column was centrifuged for 1 min to elute DNA. Alternatively, to increase DNA concentration, 30 µl Buffer EB was added and the column was incubated at room temperature for 1 min.

The column was then centrifuged as above.

2.2.5 Isolation of Plasmid DNA using Nucleospin^® Plasmid Kit

All centrifugation steps were at 13,000 rpm and at room temperature.

1) Cell lysis: 4 ml of a saturated E. coli LB culture was centrifuged for 30s and the pellet was mixed with 250 µl buffer A2 by vigorous vortexing.

250 µl buffer A2 was added and gently mixed by inverting the tube 6-8 times. The tube was then incubated at room temperature for 5 min. 300 µl buffer A3 was added and gently mixed as above.

2) Clarification: The lysate was centrifuged for 10 min and the supernatant was loaded onto the column. The colume was then centrifuged for 1 min.

3) Washing:500 µl prewarmed (at 50 °C) buffer AW was added and the column was centrifuged again for 1 min. 600 µl buffer A4 was added and the column was centrifuged for 1 min. To dry the silica membrane completely, the column was centrifuged again for 2 min.

7) Elution of DNA: 25 µl of prewarmed (at 70°C) buffer AE was added. The column was incubated for 3 min and was then centrifuged for 1 min. The elution step was repeated with further 25 µl prewarmed buffer AE, to obtain higher yield of DNA.

2.2.6 Isolation of plasmid DNA using QIAGEN Plasmid Maxi Kit

1) Cultivation of bacteria cells: A single colony was inoculated in 5 ml LB medium containing ampicillin and was incubatedfor 8h at 37°C with vigorous shaking (~250 rpm). 1 ml of this starter culture was inoculated in 500 ml LBmedium with ampicillin and was shaken as above for 16 h.

2) Cell lysis: The bacterial cells were harvested by centrifugation at 6000 rpm in a Sorvall GSA rotor at 4°C for 15 min. After the pellet was resuspended in 10 ml Buffer P1, 10 ml of Buffer P2 was added. The mixture was then gently mixed by inverting 4-6 times and was incubated at room temperature for 5 min. 10 ml of ice chilled Buffer P3 was added. The suspension was immediately mixed by inverting 4-6 times and was incubated on ice for 20 min.

3) Clarification of the lysate: The lysate was centrifuged at 13,000 rpm at 4 °C for 30 min. The supernatant containing plasmid DNA was promptly removed and centrifuged again for 15 min.

4) Column equilibration: A QIAGEN-tip 500 was equilibrated by applying 10 ml Buffer QBT. The column was then allowed to empty by gravity flow 5) DNA binding: The supernatant from step 3 was then loaded to the column

and was allowed to flow through.The QIAGEN-tip 500 was washed twice with 30 ml Buffer QC.

6) Elution and DNA precipitation: DNA was then eluted with 15 ml Buffer QF and was precipitated by adding 10.5 ml of room-temperature isopropanol.

The sample was then mixed and centrifuged immediately at 11,000 rpm in a Sorval SS-34 rotor at 4°C for 30 min. The pellet was washed with 5 ml of room-temperatured 70% ethanol and centrifuged as above for 10 min. The pellet was air-dried for 5-10 min and was redissolved in 200 µl of 10 mM Tris-HCl (pH 8,5).