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iGEM Team Heidelberg 2015

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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.

Kits

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Kit Manufacturer

QIAquick® Gel Extraction Kit (250) QIAGEN

QIAquick® PCR Purification Kit (250) QIAGEN

QIAprep® Spin Miniprep Kit (250) QIAGEN

QIAquick® PCR Purification Kit 250 QIAGEN

QIAGEN Plasmid Mini, Midi Kit (100) QIAGEN

Restriction enzymes

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Enzyme Supplier

AgeI-HF® NEB

BamHI NEB

BglII NEB

BsaI-HF® NEB

DpnI NEB

EcoRI NEB

EcoRV NEB

EcoRI-HF® NEB

HindIII-HF® NEB

NotI-HF® NEB

PacI NEB

PstI-HF® NEB

SalI-HF® NEB

SpeI-HF® NEB

XbaI NEB

Marker

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Marker Supplier

GeneRuler 100 bp DNA Ladder ThermoFisher

1 Kb Plus DNA Ladders ThermoFisher

Low Range ssRNA Ladder NEB

MagicMark™ XP Western Protein Standard ThermoFisher

Quick-Load® 2-Log DNA Ladder(0.1-10.0 kb) NEB

Quick-Load® 1 kb DNA Ladder NEB

GeneRuler Ultra Low Range DNA Ladder, ready-to-use ThermoFisher

Organisms

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Organism Supplier

BL21(DE3) E. coli DiVentura Group

NEB 5-alpha Competent E. coli (High Efficiency) NEB

One Shot® ccdB Survival™ 2 T1R Competent Cells ThermoFisher

TOP10 E. coli DiVentura Group

BY4741 yeast strain Knop Lab

HEK 293T Cell Line human Grimm Lab

HeLa Cell Line human Eils Lab

Antibiotics

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Substance Supplier

Carbenicillin disodium salt Sigma-Aldrich

Chloramphenicol ≥98% Sigma-Aldrich

Kanamycin sulfate Sigma-Aldrich

Penicillin Streptomycin (Pen Strep) ThermoFisher

Media

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Media Supplier

SOC Outgrowth Medium NEB

LB Broth Base ThermoFisher

LB Broth (Lennox) SigmaAldrich

Other enzymes

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Enzyme Supplier

DNase I (RNase-free) NEB

OneTaq® Quick-Load® 2X Master Mix with Standard Buffer NEB

Phusion Flash High-Fidelity PCR Master Mix ThermoFisher

Poly(A) Polymerase, Yeast Affymetrix

Q5® High-Fidelity DNA Polymerase NEB

T4 DNA Ligase NEB

T7 RNA Polymerase NEB

Terminal Transferase NEB

VELOCITY DNA Polymerase BIOLINE

Inorganic Pyrophosphatase ThermoFisher

RiboLock RNase Inhibitor ThermoFisher

T3 RNA Polymerase ThermoFisher

Sp6 RNA Polymerase ThermoFisher

E. coli RNA Polymerase Affymetrix

Apyrase NEB

Equipment

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Equipment Supplier

Microfuge® 18 Centrifuge Beckman Coulter

Allegra X-12R Beckman Coulter

GP 1366 Premium

HT Multitron Version 2 Infors

KTP 1750 Premium

Microfuge® 22R Centrifuge Beckman Coulter

MR Hei-Standard Heidolph Instruments

Mupid®-One Biozym

MyCyclerTM thermo cycler LabWrench

NanoDrop® ND-1000 Spectrophotometer BioCompare

PH-Meter 765 Calimatic Knick

PioneerTM PA114C TestEquipmentDepo

PioneerTM PA4101C Fisher

QBD4 GrantInstruments

Qiavac 24 Plus Qiagen

Sonoplus Gm 2070 Bandelin

T100 Therma Cycler BIO-RAD

Thermomixer comfort Eppondorf

Ultrospec10 GelLifesciences

Vacuhand control Vacuubrand

VORTEXGENIE 2 ScientificIndustries

Backbones

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Backbones Supplier

pSB1C3 iGEM registry

pSB1A2 iGEM registry

pIRES-U6-EGFP Grimm Lab

p415 GPD Knop Lab

p413 GPD Knop Lab

Buffer

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Name Detail

Phusion HF Buffer NEB

Phusion GC Buffer NEB

10x BSA Buffer 440 mM Tris HCl 8.1; 40 µg/ µL BSA; 2% v/v Glycerol; 300 mM KCl; 200 mM MgCl2

2x Loading Dye 95 % v/v Formamid; 5 % 10 x TBE Buffer (Roth); 1 Tip of a spatula of Bromphenol Blue and Xylenxylanol

10x Transcription Buffer 100 mM Spermidine 400 mM Tris HCl pH 8.1; 100 mM Spermidine; 220 mM MgCl2; 0.1 % Triton X

10x Transcription Buffer 10 mM Spermidine 400 mM Tris HCl pH 8.1; 10 mM Spermidine; 220 mM MgCl2; 0.1 % Triton X

10x Transcription Buffer w/o Spermidine 400 mM Tris HCl pH 8.1; 220 mM MgCl2; 0.1 % Triton X

10x Tris HCl/ KCl Buffer for in vitro transcription 440 mM Tris HCl pH 8.1; 300 mM KCl; 200 mM MgCl2

10x TBE Tris HCl; EDTA; Boric Acid

2x Renaturing Buffer 40 mM HEPES, pH 7.5; 125 mM KCl

DFHBI Solution 198 µl DMSO; 1 mg DFHBI