Journal: Current Protocols
Article Title: Ligase‐Catalyzed Transcription and Reverse Transcription of XNA by T3 DNA Ligase
doi: 10.1002/cpz1.70293
Figure Lengend Snippet: Example of PAGE analysis of PCR amplification of LOOPER‐RT product. Lane 1, Quick‐Load Purple Low Molecular Weight DNA Ladder; lane 2, no‐template control; lane 3, PCR amplification of LOOPER‐RT product using RTL‐3p and RTL‐T7p1 primers after 19 cycles. The full‐length amplicon is indicated.
Article Snippet: Cycle Pure Kit (Omega Bio‐tek, D6492‐02) 100% ethanol (required for Omega kit) 2× gel loading buffer with dyes (Invitrogen, cat. no. AM8547) Precast 10% polyacrylamide TBE‐urea gel (Bio‐Rad, cat. no. 4566033) 1× TBE buffer (see recipe) Low‐molecular‐weight DNA ladder: ssDNA ladder, 20/100 (IDT, cat. no. 51‐05‐15‐02) Quick‐Load Purple Low Molecular Weight DNA Ladder (NEB, cat. no. N0557S) DNA stain: ethidium bromide or SYBR Safe (Life Technologies) 3 M NaCl (see recipe) 10 μM RTL_T7p1 (Integrated DNA Technologies; Table ) 10 μM RTL‐3P (Integrated DNA Technologies; Table ) 1 mM degenerate 5‐phosphorylated DNA trinucleotide library [(51 mM‐P) (dN)(dN)(dN), Dharmacon, Horizon] in H 2 O 2× Q5 High‐Fidelity Master Mix (NEB M0492S) 0.2‐ml PCR tubes (Fisher, cat. no. 14230205) Vortex mixer (Scientific Industries, SI‐0236) Minicentrifuge (Corning, cat. no. 6770) Microcentrifuge (Eppendorf, cat. no. 5405000441) 1.5‐ml microcentrifuge tubes (VWR, cat. no. 87003‐294) Vacuum concentrator (SpeedVac SPD120, Thermo Scientific) Thermal cycler (Bio‐Rad, cat. no. 1861096) Vertical electrophoresis system (Bio‐Rad, cat. no. 1658004) Electrophoresis power supply (Bio‐Rad, cat. no. 1645050) Incubator (Corning, cat. no. 6800), preheated to 55°C Gel imaging system (Gel Doc XR+ Gel Documentation System, Bio‐Rad) Blue light transilluminator (BIO‐HELIX, cat. no. BP001CU) Aluminum foil or dark cabinet or drawer x‐tracta gel extraction tool (Sigma‐Aldrich, cat. no. Z722390) Centri‐Sep Spin Column (Princeton Separations, cat. no. CS‐900)
Techniques: Amplification, Molecular Weight, Control
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