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RNA Scaffold Overview

The results covered in this section are of the experiments overviewed in the RNA Scaffold Design section.

RNA Scaffold Data

Fig. 1. 1 L culture incubated at 37oC till 0.5 nm optical density after inoculation with 5 mL of overnight for WT PUF-PIN and 6-2/7-2 PUF-PIN. 2 mM IPTG induction for 2 hours. 200 mL cultures incubated at 37oC till 1 nm optical density after inoculation with 2 mL of overnight for WT PUF-αGFP cultures. 2 mM IPTG induction for various hours. SDS-PAGE 6% native gel stained with Coomassie Brilliant Blue for 1 hr. and destained with H20 for 1 hr.



Fig. 2. 1 L culture incubated at 37oC till 0.5 nm optical density after inoculation with 5 mL of overnight. 2 mM IPTG induction for 2 hours. His-Tag Ni-NTA purification, centrifuged with Millipore 30kDa cutoff ultracentrifuge tubes. SDS-PAGE 6% native gel stained with Coomassie Brilliant Blue for 1 hr. and destained with H20 for 1 hr.


Fig. 3. 1 L culture incubated at 37oC till 0.5 nm optical density after inoculation with 5 mL of overnight. 2 mM IPTG induction for 2 hours. His-Tag Ni-NTA purification, centrifuged with Millipore 30kDa cutoff ultracentrifuge tubes. SDS-PAGE 6% native gel stained with Coomassie Brilliant Blue for 1 hr. and destained with H20 for 1 hr.




Fig. 4. BCA analysis of WT & 6-2/7-2 PUF-PIN proteins to determine concentration (uM). 2 mg/mL BSA concentration used. Click on Fig. 4. to view it in a higher resolution.



Fig. 5. 10 well 10% 1mm urea denaturing acrylamide gel, post 2μg/mL EtBr staining for 20 min., destaining for 20 min. 1X TBE buffer, 120V for 55 min. In-Vitro Transcription with MEGAscript® T7 Kit (Invitrogen)



Fig. 6. 10 well 10% 1mm urea denaturing acrylamide gel, post 2μg/mL EtBr staining for 20 min., destaining for 20 min. 1X TBE buffer, 120V for 55 min. First 4 wells include 1 μL of 30 mM MnCl2 ions, last 4 wells include 1 μL of 30 mM MgCl2 ions. RNA samples were denatured for 3 min. at 95oC and then let to fold at 4oC for 5 min. Addition of 1 μL of annealing buffer (EDTA/Tris) before addition of protein. 30 min. incubation time with protein at 37oC and then addition of 2X 80% formamide/EDTA to stop the reaction. 66 nM concentration of RNA, 305 nM concentration of WT PUF-PIN, and 45 nM concentration of 6-2/7-2 PUF-PIN used.

PUF Tethering Data

Fig 1. 50mL 6 well 1% agarose gel with 5 μL of 10 mg/mL EtBr, 1X TAE buffer, 130V for 25 min. PCR amplifications done in GC Buffer, 68.7oC 30 sec. annealing time, 35 cycles for amplification and 10 cycles for tethering. PUF and cCFP (lanes 2 & 6) were first amplified and then used in the tethering reaction in lane 3.



Fig 2. 50mL 6 well 1% agarose gel with 5 μL of 10 mg/mL EtBr, 1X TAE buffer, 130V for 25 min. PCR amplifications done in HF Buffer, 68.7oC 30 sec. annealing time, 10 cycles of PCR.



Fig 3. 50mL 10 well 1% agarose gel with 5 μL of 10 mg/mL EtBr, 1X TAE buffer, 130V for 25 min. PCR amplifications done in GC Buffer, 1 min. annealing time, 10 cycles of PCR, half the volume of primers used (0.5 μL instead of 1 μL).



Fig 4. 50mL 6 well 1% agarose gel with 5 μL of 10 mg/mL EtBr, 1X TAE buffer, 130V for 25 min. PCR amplifications done in GC Buffer, 68.7oC 30 sec. annealing time, 25 cycles of PCR.

Conclusion