Difference between revisions of "Template:Heidelberg/pages/methods"

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Revision as of 17:18, 17 September 2015

Methods
Extension PCR Extension PCR was performed using Phusion Flash master mix with equimolar amounts of overlapping oligo DNA and 5 % DMSO.
In vitro Transcription dsDNA was transcribed into sRNA with T7 RNA polymerase in transcription buffer in the presence of 4 mM ATP, GTP, CTP, UTP each, 10 mM DTT and 5 % DMSO. Reaction was incubated at 37 °C for ~3 h. Then DNase I was added an the reaction was further incubated at 37 °C for 20 min. RNA of interested was purified in 10 % denaturing PAGE. Band were visualized by UV shadowing and appropriate band was excised and eluted using 0.3 M NaAc pH 5.5.
3’ Modifiction of sRNA Various amounts of sRNA (1-20 µM) were incubated with modified NTPs (150-400 µM) (Biotin-, Alkyne-, Azide-NTP) using either yeast Poly A Polymerase (PAP), affimetrix or Terminale dinucleotidyl Transferase (TdT), NEB in supplied buffers. Reaction was precipitated. Success was check via CuAAC.
Precipitation of nucleic acids RNA/DNA was precipitated in the presence of 0.3 M NaAc pH 5.5 at -20 °C by the addition of 2.5 volumes of -20 °C cold EtOH. After storing sample at –20 °C for at least 1.5 h is was centrifuged at 14,000 g. Supernatant was removed and pellet was dissolved in H2O. The concentration of the sample was determined using a NanoDrop and calculated using the extinction coefficient from idt oligoanalyzer.
Copper-catalyzed 1,3-dipolar azide-alkyne cycloaddition (CuAAC) Copper click reaction was performed as described in Winz, 2012. Success was check on denaturing PAGE that was first scanned in appropriate fluorescent mode and after staining with SYBR Gold in SYBR Gold mode on a Typhoon Scanner, GE.
Initial DNAzyme Activity DNAzyme activity was initially tested by incubating 0.5- 5 µM DNAzyme with 200nM Substrate in DNAzyme buffer at 37 °C for 1.5 h. Then 2 µL (to 25 µL reaction) were added and DNAzyme was digested for 20 min at 37 °C. Reaction was separated on 20 % denaturing PAGE and visualized with SYBR Gold.
Denaturing Polyacrylamid gel electrophorese (PAGE) Polyacrylamide gels were prepared using Rotiphorese Sequencing Gel System according to manual. Gel were run in TBE buffer. RNA or DNA was visualized by UV shadowing or SYBR Gold stain and scanning with a Typhoon Scanner, GE.
PCR PCR was performed using Phusion Flash Master Mix (Thermo Scientific), OneTaq® Quick-Load® 2X Master Mix (NEB), Q5® High-Fidelity 2X Master Mix (NEB) or Velocity DNA Polymerase (Bioline) according to manufacturer’s protocol.
PCR Purification PCR was purified using QIAquick PCR Purification kit from Qiagen according to manufacturer’s protocol.
Gel Extraction To extract DNA from agarose gel sample was run on a 0.8 % gel in TAE buffer. DNA was visualized under UV using EtBr. Suitable bands were excised and DNA was extracted using QIAquick Gel Extraction kit according to manufacturer’s protocol.
Agarose gel electrophorese To check the size of DNA fragments or to purify DNA samples were run on a 0.8-4 % agarose gel. To prepare the gel 0.8-4 % (w/v) agarose were heated in TAE or TBE buffer in the microwave. Gel was poured containing ethidium bromide or Roti Gel Stain and visualized by UV light.
Digest of DNA with restriction enzymes DNA was incubated with restriction enzyme in appropriate buffer and incubated as described in the manual. If necessary enzymes were heat inactivated and afterwards purified by precipitation or PCR purification kit.
Initial DNAzyme Activity DNAzyme activity was initially tested by incubating 0.5- 5 µM DNAzyme with 200nM Substrate in DNAzyme buffer at 37 °C for 1.5 h. Then 2 µL (to 25 µL reaction) were added and DNAzyme was digested for 20 min at 37 °C. Reaction was separated on 20 % denaturing PAGE and visualized with SYBR Gold.
Ligation DNA with matching sticky ends were ligated using T4 DNA Ligase in supplied buffer.
Cryostock of E. coli strain 500 µL of oN culture was mixed with 500 µL of autoclaved 40 % glycerol and stored at -80 °C.
Plasmid prep Plasmids were purified from liquid cultures using QIAprep Mini-, Midi or Maxiprep according to manufacturer’s protocol. Concentration was determined with a NanoDrop.