Ptc 225 peltier thermal cycler

Total RNA (5 ng) was reverse-transcribed using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) and the miRNA-specific reverse-transcription primers provided with the TaqMan MicroRNA Assay (Applied Biosystems). For the reverse transcription, a PTC-225 Peltier Thermal Cycler (MJ Research Inc., Waltham, Massachusetts) was used. The reaction was performed at 16°C for 30 min; 42°C for 30 min, and 85°C for 5 min. The obtained miRNA-specific cDNA was amplified using the TaqMan Universal PCR master mix II (Applied Biosystems) and the respective specific probe provided in the TaqMan Small RNA Assay (Applied Biosystems). PCR was performed using a CFX-96TOUCH (BIO-RAD). Amplification was performed at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. U6 small nuclear RNA was used as an endogenous control. The fold change in the miRNA level was calculated by the log⁡2 scale of the equation 2-ΔΔCt, where ΔCt = Ct miRNA-Ct U6 and ΔΔCt = ΔCt TMH samples − ΔCt DM samples [15 (link)].

Three published 18S rRNA gene PCR methods specific for N. perurans [31 (link)], N. pemaquidensis [32 ] and N. branchiphila [33 (link)] were used to detect and identify the species of amoebae in the culture.
Genomic DNA was extracted from an aliquot of the established in vitro culture, containing 5 × 10³N. perurans cells, using the EZ1 DNA tissue Kit and an EZ1 extraction robot (Qiagen, Manchester, UK) following the manufacturer's protocol.
PCR reactions were performed in a 50 μL reaction volume consisting of 1x GoTaq flexi buffer (Promega, UK), 2.5 mM MgCl₂, 0.25 mM each dNTP, 50 pmol of the forward and reverse primers, 1.25 units of GoTaq^® DNA Polymerase (Promega, UK) and 2.5 μL of the extracted DNA. The reaction mix was overlaid with mineral oil and after an initial denaturing step (5 min at 95 °C), was subjected to 35 temperature cycles (1 min at 95 °C, 1 min at 55 °C and 1 min at 72 °C) in a PTC-225 Peltier thermal cycler (MJ Research, Canada) followed by a final extension step of 10 min at 72 °C. PCR products were visualised on 2% agarose gels stained with ethidium bromide and purified using GENECLEAN^® (Anachem, UK). Both DNA strands were sequenced using the ABI PRISM™ dye terminator cycle sequencing kit (Perkin Elmer, UK) on an ABI 310 genetic analyser. Sequence similarity searches were conducted using blastn [34 (link)] and the NCBI nucleotide database.

Reaction mixtures consisted of 10 ng of total genomic DNA, 50 μmols each of PCR primers, 2.5 mM dNTPs, 2.5 mM MgCl₂, 1× reaction buffer, and 0.25 U of Taq DNA Polymerase (Qiagen) in a final volume of 10 μl. The thermal profile for each primer pair was tested in gradient thermocycler (PTC-225 Peltier Thermal Cycler; MJ Research). The following profile: [95 °C–7′] [94 °C–10″; X °C–30″; 72 °C–90″] ×45 [72 °C–10′] [5 °C ∞], where X ranged from 48 to 67 °C (Supplementary Table 1) depending on the primer pair, was used. The PCR products were separated on 1.2 % agarose gels in TBE buffer at 5 V/cm for 1 h.
To distinguish DArT markers from their PCR-based counterparts, their names were extended with ‘c’ (i.e., rPt-508078 vs. rPt-508078c).

Unpurified dehydrated staple oligonucleotides were purchased from Integrated DNA Technologies (IDT) at 10 nmol scale. Each unpurified staple strand was rehydrated at ~100 μM in Milli-Q water and then equal volumes pooled together. The p8064 scaffold strand was produced from M13 phage replication in Escherichia coli. The DNA-origami seed was folded with 10 nM p8064 scaffold and ~100 nM of each unpurified staple strand in 1X TE buffer (5 mM Tris and 1 mM EDTA) containing 6 mM MgCl₂. The reaction was incubated on a PTC-225 Peltier Thermal Cycler (MJ Research) with the following temperature program: 90 °C for 2 min, cool to 55 °C and decrease to 50 °C over 18 h by decreasing the temperature at a rate of −1 °C/3 h, and then holding the temperature at 4 °C thereafter. The folded reaction was analyzed on an agarose gel and by observation with TEM. The folded seed was stored at 4 °C in the raw folding mixture. The seed was noted to remain stable in 4 °C storage for up to a year and was used directly from the raw folding reaction in slat assembly experiments. Staple and scaffold DNA sequences are reported in Supplementary Data S1.

Bacterial identification was carried through 16s rRNA sequencing. Bacterial genomic DNA was isolated using the Insta GeneTM Matrix genomic DNA isolation kit Catalog # 732-6030. Using 16s rRNA universal primers gene fragment was amplified using MJ Research PTC-225 Peltier Thermal Cycler. The sequence obtained is deposited in the NCBI gene bank using the tool Sequin.