3 Materials and methods
3.1 Materials .1 Chemicals
3.2.1 Molecular biology methods
3.2.1.1 Concentration determination of nucleic acids
To determine the concentration of nucleic acids, the extinction in aqueous solution was measured at a wavelength of 260 nm in comparison to a reference (buffer). Following equations were used to calculate the concentration (Sambrook et al., 1989):
1 OD260 = 50 µg/ml double stranded DNA = 0.15 mM (in nucleotides) 1 OD260 = 33 µg/ml single stranded DNA = 0.10 mM (in nucleotides) 1 OD260 = 40 µg/ml single stranded RNA = 0.11 mM (in nucleotides)
The concentration of α-[32P]UTP-labeled RNA was determined by the ratio of α-[32P]UTP and
31P-UTP, the isotope concentration of α-[32P]UTP, the absolute number of uridines in the transcript and an instrument constant using the following equations:
mixing ratio P UTP
α P UTP
isotope concentration Ci mmol
mixing ratio # Uridines radioactivity Ci mmol
speci ic activity dpm
mmol radioactivity Ci
mmol 2.2 · 10 dpm Ci speci ic activity cpm
mmol speci ic activity dpm mmol 2
3.2.1.2 Phenol-Chloroform-Isoamylalcohol (PCI) extraction
Phenol-chloroform-isoamylalcohol (PCI) extraction was used to purify and separate proteins and nucleic acids. The sample was mixed with 1 vol. of PCI and 1 µl of 10 µg/µl glycogen and vigorously agitated on a vortex. Aqueous and organic phases were separated by centrifugation for 5 min at room temperature (13,000 rpm). The aqueous phase, containing nucleic acids, was transferred to a new tube. Proteins (organic phase) were precipitated with 5 vol. 100 % (v/v) acetone at -20 °C for at least 2 h. Nucleic acids (aqueous phase) were further purified by addition of 1 vol. of chloroform. After mixing and centrifugation as above,
the nucleic acids were precipitated from the aqueous phase in 3 vol. of 100 % (v/v) ethanol and 1/10 vol. of 0.3 M sodium acetate pH 5.3 for at least 2 h at -20 °C. Precipitated proteins or nucleic acids were collected by centrifugation for 30 min at 4 °C (13,000 rpm). Proteins or nucleic acids were washed with 80 % (v/v) ethanol, collected by centrifugation (see above) and dried in a vacuum centrifuge.
Phenol-Chloroform-Isoamylalcohol (PCI):
50 % (v/v) Phenol 48 % (v/v) Chloroform 2 % (v/v) Isoamylalcohol
3.2.1.3 Agarose gel electrophoresis of DNA
Agarose gel electrophoresis was used to analyze and purify DNA. Gels were prepared with 1 % (w/v) agarose and 0.5 µg/ml ethidium bromide to visualize DNA by UV light. DNA samples were diluted with 5 × DNA loading dye and allowed to migrate horizontally at 120 V using 1 × TBE as running buffer until the dye migrated through the gel. DNA was visualized under a UV light at a wavelength of 365 nm.
Gel solution: DNA loading dye:
1 % (w/v) Agarose 30 % (w/v) Glycerol
1 × TBE 0.25 % (w/v) Bromophenol blue
0.5 µg/ml Ethidium bromide
3.2.1.4 In vitro transcription
Pre-mRNAs were synthesized by in vitro transcription using SP6 polymerase and linearized plasmid DNA as template. The reaction was incubated at 40 °C for 3-4 h. Template DNA was subsequently digested with 1U RQ1 DNAse/µg template DNA at 37 °C for 20 min. RNA transcripts were purified by gel purification using 5 % polyacrylamide gels containing 8 M urea. Unlabeled RNA was visualized by UV-shadowing (254 nm) and α-[32P]UTP-labeled RNA was visualized by exposure to an X-ray film (1 min). Bands were excised from the gel and extracted by incubation with RNA extraction buffer overnight. Extracted RNA was further purified by PCI extraction and ethanol precipitation. The purified RNA was resuspended with RNAse-free water.
Preparation of capped PM5-pre-mRNA:
α-[32P]UTP -labeled non-labeled
5 × transcription buffer 10.00 µl 30.00 µl
0.1 M ATP 3.75 µl 11.25 µl
0.1 M UTP 3.75 µl 11.25 µl
0.1 M CTP 3.75 µl 11.25 µl
0.01 M GTP 6.50 µl 19.50 µl
152 mM m7GpppG cap 1.64 µl 4.92 µl
32P-αUTP (3000 Ci/mmol) 5.00 µl -
1M MgCl2 1.05 µl 3.15 µl
1M DTT 0.50 µl 1.50 µl
10 mg/ml BSA 0.50 µl 1.50 µl
40 U/µl RNAsin 1.25 µl 3.75 µl
SP6 polymerase 5.33 µl 16.00 µl
DNA template 5.00 µg 15.00 µg
The volume was adjusted to (RNAse-free water)
50 µl 150 µl
8 M urea-5 % Polyacrylamide Gel Solution (100 ml):
12.5 ml Rotiphorese Gel 40
42 g Urea
10 ml 10 × TBE
Adjust to 100 ml (RNAse free water) Per 100 ml gel solution:
10 µl TEMED
100 µl 10 % (w/v) APS
3.2.1.5 DNA amplification
100 µl chemical competent dH5α cells were transfected with 100 ng of plasmid DNA by the heat shock method according to standard protocols (Sambrook et al., 1989). To this end, competent bacteria were thawed on ice and mixed with plasmid DNA. After incubation on ice for 30 min, cells were heat shocked at 42°C for 90 s and subsequently cooled on ice for 2 min. 1 ml of LB medium was added followed by incubation at 37 °C for 1 h. To select transformed bacteria, cells were plated on agar plates containing the appropriate antibiotic and incubated at 37 °C overnight. Single clones were selected and grown at 37 °C in LB medium. DNA was recovered using QIAprep Spin Miniprep kit or Invitrogen PureLink HiPure Plasmid Filter Maxiprep kit.
3.2.1.6 Restriction digestion of DNA
Restriction digestion was used to generate desired ends of template DNA for in vitro transcription from plasmid DNA. 200 µg of plasmid DNA, 100 U of BamHI, 140 µl of 10 × Buffer 4 were adjusted to a final volume of 1400 µl with RNAse-free water. The mixture was incubated at 37 °C overnight and linearized DNA was recovered by PCI extraction.
Linearization was assayed by agarose gel electrophoresis.
3.2.1.7 Proteinase K digestion
Proteinase K digestion was performed to improve RNA recovery from splicing reactions.
125 µl of proteinase K mix were added to one 20 µl aliquot of splice reaction followed by incubation for 1h at 37 °C. The volume was adjusted to a final volume of 200 µl with 35 µl 1 × D- buffer and 20 µl of 10 % (v/v) SDS. The RNA was recovered by PCI extraction and ethanol precipitation.
Proteinase K mix:
62.5 µl 2 × PK buffer 1 µl 10 mg/ml Glycogen 57.5 µl RNAse-free water 4 µl 10 mg/ml Proteinase K
3.2.1.8 Denaturing polyacrylamide gel electrophoresis
Denaturing gel electrophoresis was used to analyze snRNA and pre-mRNA splicing products. RNA samples were dissolved in RNA sample buffer, heated for 3-5 min at 96 °C and loaded onto 0.5 mm thick 10 % polyacrylamide gels (37.5:1 acrylamide to bis-acrylamide ratio) containing 8 M urea. Electrophoresis was performed vertically at 20 W using 1 × TBE as running buffer.
8 M urea-10 % Polyacrylamide RNA Sample Buffer:
Gel Solution (100 ml):
25.0 ml Rotiphorese Gel 40 80 % (w/v) Formamide
42 g Urea 1 mM EDTA
10 ml 10 × TBE 0.05 % (w/v) Bromophenol blue
Adjust to 100 ml (RNAse free water) 0.05 % (w/v) Xylene cyanol Per 100 ml gel solution:
10 µl TEMED
100 µl 10 % (w/v) APS
3.2.1.9 Silver staining of RNA
RNA was visualized by silver staining according to Schoenle et al., 1984. Briefly, RNA was fixed in 40 % (v/v) methanol/10 % (v/v) acetic acid for at least 30 min and washed twice with 10 % (v/v) ethanol/5 % (v/v) acetic acid for 15 min. The gel was briefly rinsed with water and incubated with 12 mM silver nitrate for 30 min. After 3 short rinsings with water, the gel was incubated with 280 mM sodium carbonate/0.0185 % (v/v) formaldehyde until the desired staining intensity was reached. Staining was then stopped immediately with 5 % (v/v) acetic acid.
3.2.1.10 Native gel electrophoresis of RNA complexes
Splicing complex formation was analyzed by native gel electrophoresis. 0.7 µl Heparin (5 mg/ml, final concentration 170 mg/ml) was added to 20 µl aliquots of splicing complexes and heated for 1 min at 30 °C. Samples were diluted with 5 × native loading dye and loaded onto 1.5 % agarose gels. Electrophoresis was performed horizontally at 40 V for 15 h using 0.5 % TBE as running buffer. The gels were fixed with 10 % (v/v) acetic acid/10 % (v/v) methanol for 30 min and dried under vacuum at 60 °C for 4.5 h. Radioactively labeled RNA was visualized by exposing the dried gels to phosphorimager screens for approx. 1 hr and scanning using Typhoon PhosphorImager.
Agarose Gel Solution: Native loading dye:
1.5 % (v/v) Low Melting Point Agarose 1 × TBE
0.5 × TBE 30 % Glycerol
0.02 % Bromophenol blue
3.2.1.11 Generation of pre-mRNA mutants (deletion of 5’ss and BPS)
5’splice site (5’ss)-, branch point site (BPS)- and BPS-ACTGA-deleted PM5 pre-mRNA was generated as described before (Dönmez, 2006; see also Figure 3.1 for an overview). PCR primers were used to exclude the 5’SS or BPS region within the PM5 plasmid. The PCR product was purified by agarose gel electrophoresis and recovered using the NucleoSpin Extract II kit (Machery-Nagel). The linear plasmid was kinased with T4 ploynucleotide kinase (T4 PNK) in the presence of 1mM ATP. The reaction was incubated for 1 h at 37 °C and the kinased plasmid was purified using the NucleoSpin Extract II kit. The plasmid was then ligated with T4 DNA ligase for 30 min at 37 °C followed by incubation at 16 °C overnight. The ligase was deactivated by incubation for 10 min at 65 °C. The ligated plasmid was purified by ethanol precipitation, dissolved in water and used for transformation of dH5α cells. Amplified
DNA was isolated using the QIAprep Spin Miniprep kit and cloned plasmids were sequenced (Seqlab Sequence Laboratories Göttingen GmbH).
PCR reaction: (100 µl) PCR program:
0.5 µl PM5 plasmid (20 µg/ml) 1 µl Turbo Pfu
1.6 µl dNTPs (25 mM each) 1 µl forward primer (100 µM) 1 µl reverse primer (100 µM) 10 µl cloned Pfu buffer 7.5 µl DMSO
77.4 µl H2O
Figure 3.1: Generation of 5’ss, BPS and BPS-ACTGA deleted PM5 pre-mRNA. PCR primers were used to exclude the sequence to be deleted. After purification of the linear PCR product, the DNA was kinased using T4 PNK and subsequently ligazed with T4 DNA Ligase. The obtained plasmid DNA does not include the deleted nucleotide sequence.
3.2.2 Protein biochemical methods