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From 2012.igem.org

Revision as of 22:16, 3 October 2012 (view source) Bchen26 (Talk | contribs) ← Older edit		Revision as of 22:19, 3 October 2012 (view source) Bchen26 (Talk | contribs) Newer edit →
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-	<img src="https://static.igem.org/mediawiki/2012/d/d4/Tether1.jpg" height=50% width=50%></center>	+	<img src="https://static.igem.org/mediawiki/2012/f/f5/IGB_2012-07-24_14hr_22min_PUF(for_tethering)%2C_PUF-cCFP%2C_cCFP(for_Split-CFP)%2C_cCFP(for_tethering).jpg"></center>
	<br/><b>Fig 1.</b> 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.		<br/><b>Fig 1.</b> 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.
	<br/><br/><br/>		<br/><br/><br/>
	<center>		<center>
-	<img src="https://static.igem.org/mediawiki/2012/b/b1/Tether2.jpg" height=50% width=50%></center>	+	<img src="https://static.igem.org/mediawiki/2012/e/e9/IGB_2012-07-25_14hr_44min_PUF-cCFP_Tether%2C_4clsts_colonypcr.jpg"></center>
	<br/><b>Fig 2.</b>		<br/><b>Fig 2.</b>
	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.		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.
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-	<img src="https://static.igem.org/mediawiki/2012/c/cf/Tether3.jpg" height=50% width=50%></center>	+	<img src="https://static.igem.org/mediawiki/2012/2/27/IGB_2012-07-26_15hr_27min_cCFP-pETDuet-1_Digestion%2C_PUF-cCFP_tether_gradient.jpg" ></center>
	<br/><b>Fig 3.</b>		<br/><b>Fig 3.</b>
	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).		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).
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	<center>		<center>
-	<img src="https://static.igem.org/mediawiki/2012/b/b6/Tether4.jpg" height=50% width=50%></center>	+	<img src="https://static.igem.org/mediawiki/2012/c/c6/IGB_2012-07-27_10hr_41min_PUF%2C_cCFP_Amplification_for_tethering.jpg"></center>
	<br/><b>Fig 4.</b>		<br/><b>Fig 4.</b>
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	<center>		<center>
-	<img src="https://static.igem.org/mediawiki/2012/f/f5/Tether5.jpg" height=50% width=50%></center>	+	<img src="https://static.igem.org/mediawiki/2012/2/27/IGB_2012-07-26_15hr_27min_cCFP-pETDuet-1_Digestion%2C_PUF-cCFP_tether_gradient.jpg"></center>
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-	<br/><b>Fig 5.</b>	+	<br/>
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	<div id="scaffold3" style="display:none">		<div id="scaffold3" style="display:none">

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Revision as of 22:19, 3 October 2012

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.



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.


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.




Fig. 4. In-Vitro Transcription with MEGAscript® T7 Kit (Invitrogen)



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. 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 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.