Linker PCR was used to verify the complexity of the generated transposon mutant library. DNA was extracted (Gram positive DNA extraction Epicentre – lysing the cells with Ready-Lyse Lysozyme over night) from the transposon mutant pool in addition to DNA from 15 randomly isolated colonies (BHI plates containing 5 mg/l erythromycin) representing 15 random transposon mutants from the library. The DNA was digested with RsaI (Promega) and purified using a Minielute PCR purification kit (Qiagen). Adaptor molecules were made by mixing a 1∶1 ratio (100 µM) of oligo 254 and 256 (see Table 1 ), denatured at 95°C for 3 min. in annealing buffer (10× annealing buffer = 100 mM Tris pH8, 500 mM NaCl, 10 mM EDTA) and annealed at room temperature for 1 hour (store at −20°C). Adaptors and digested DNA were ligated using a Quick DNA Ligase (New England Biolabs) followed by purification using a PCR purification kit (Qiagen). A PCR with primers ForwardTnL and reverse primer 258 (see Table 1 ) and Hotstar taq polymerase (Qiagen) was conducted with the following conditions: Hot-start 15 min at 95°C, 30 cycles of denaturation for 45 sec at 94°C, annealing 1 min at 55°C and elongation for 2 min at 72°C and a final elongation for 5 min at 72°C. The PCR products were visualised on a 2% NuSieve GTG Agarose gel (Lonza) (3 hours, 100 volts).
Nusieve gtg agarose gel
Manufactured by Lonza
Sourced in Switzerland
NuSieve GTG Agarose gel is a laboratory product used for gel electrophoresis applications. It serves as a separation medium for the analysis and purification of nucleic acids, such as DNA and RNA.
Automatically generated - may contain errors
Lab products found in correlation
Hotstar taq polymerase, by Qiagen (1 mentions)
Pcr purification kit, by Qiagen (1 mentions)
Minielute pcr purification kit, by Qiagen (1 mentions)
Quick dna ligase, by New England Biolabs (1 mentions)
Loading dye, by Thermo Fisher Scientific (1 mentions)
Chemidoc touch, by Bio-Rad (1 mentions)
High pure pcr product purification kit, by Roche (1 mentions)
Takara ex taq hot start version, by Takara Bio (1 mentions)
Takara pcr thermal cycler dice, by Takara Bio (1 mentions)
Revertra ace qpcr rt master mix with gdna remover, by Toyobo (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Taq polymerase, by Thermo Fisher Scientific (1 mentions)
Gelred nucleic acid gel stain, by Biotium (1 mentions)
Hinfi enzyme, by New England Biolabs (1 mentions)
4 protocols using nusieve gtg agarose gel
1
Verifying Transposon Mutant Library Complexity
2
miRNA Isolation and Visualization
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The sCA-miRNA pellet was dissolved in 100 μL of 0.02 M EDTA. The collected miRNA sample was mixed with loading dye (Thermo Fisher Scientific, Waltham, MA, USA) and loaded in a 4.5% NuSieve GTG agarose gel (Lonza, Basel, Switzerland). Imaging was performed using ChemiDoc Touch (Bio-rad, Hercules, CA, USA).
3
Tumor Mutation Profiling by Sanger Sequencing
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Tumor RNA samples were reverse‐transcribed with ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan) according to the manufacturer's instructions. Primer3Plus (http://primer3plus.com/ ) was used to design the primer pairs to amplify the region ranging from 200 to 600 bp in size containing each mutation (Table S1 ). All primers were synthesized by Sigma‐Aldrich Japan (Tokyo, Japan). TaKaRa Ex Taq Hot Start Version (TaKaRa Bio, Kusatsu, Japan) was used for PCR amplification according to the manufacturer's protocol. The following steps were carried out using a TaKaRa PCR Thermal Cycler Dice (TaKaRa Bio): 94°C for 3 min, 40 cycles of 94°C for 30 s, and 60°C for 30 s and 72°C for 1 min, followed by incubation at 72°C for 3 min. The PCR products were electrophoresed and amplicons of the expected size excised from NuSieve GTG agarose gels (Lonza, Basel, Switzerland), and then purified using High Pure PCR Product Purification Kits (Roche, Basel, Switzerland). Amplicons were Sanger sequenced with the primer used for PCR amplification (FASMAC, Atsugi, Japan).
4
Genotyping TLR4 T399I Polymorphism
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Genomic DNA was extracted from 5 mL of peripheral whole-venous blood using a salting out procedure, as previously described [24 (link)], and quantified by NanoDrop 2000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA). T399I polymorphism (+1196C/T transition; rs4986791) was typed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Briefly, the DNA region comprehending the polymorphism was amplified by PCR by using the TLR4 T399I-forward: 5′-GGTTGCTGTTCTCAAAGTGATTTTGGGAGAA-3′ and TLR4 T399I-reverse: 5′-ACCTGAAGACTGGAGAGTGAGTTAAATGCT-3′ primers. Amplifications were carried out in a final volume of 30 μL including 200 ng of DNA template, primers at 500 nM and 1 U of Taq polymerase (Invitrogen, Carlsbad, CA, USA). PCR conditions were as follows: denaturation for 4 min at 95 °C, 35 cycles of 45 sec at 95 °C, 45 sec at 67 °C and 90 sec at 72 °C, followed by 5 min at 72 °C. The specific amplificated band of 406 bp was digested by a HinfI enzyme (New England Biolabs, Ipswich, MA, USA), following the manufacturer’s instructions. PCR-RFLP products were separated upon digestion in 3% NuSieve GTG agarose gels (Lonza Rockland, Rockland, ME, USA) with GelRed Nucleic Acid Gel Stain (Biotium, Fremont, CA, USA) and visualized under Ultraviolet (UV) light by using a ChemiDoc XRS analyzer (Bio-Rad, Hercules, CA, USA).
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