Biomod/2014/UCR/Breaking RNA/Acknowledgements: Difference between revisions

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<h3><font size="4.5">2.3 Bound Aptamer-Kleptamer Interactions: Gel Electrophoresis</font></h3>
<h3><font size="4.5">2.3 Bound Aptamer-Kleptamer Interactions: Gel Electrophoresis</font></h3>
The concentration of R1 and K1 used are 1 &mu;M and 2 &mu;M and the T7 RNA Polymerase volume used was 1 &mu;L. The total volume in each well was 7 &mu;L. R1 and SP6 RNAP were mixed together and incubated for 20 minutes at 30°C. Next, K1 was added to the mixture and the solution was incubated for another 10 minutes at 30°C. A list of the components in each well is given below:
For the first gel, the concentration of R1 and K1 used are 1 &mu;M and 2 &mu;M and the T7 RNA Polymerase volume used was 1 &mu;L. The total volume in each well was 7 &mu;L. R1 and SP6 RNAP were mixed together and incubated for 20 minutes at 30°C. Next, K1 was added to the mixture and the solution was incubated for another 10 minutes at 30°C. A list of the components in each well is given below:
::{| border = "1" class="wikitable" style="margin:auto;"
::{| border = "1" class="wikitable" style="margin:auto;"
! scope="col" | Lane
! scope="col" | Lane
Line 131: Line 131:
|}
|}


For the second gel, the concentrations of R1 and R4 used were both 0.7 &mu;L and the SP6 RNA Polymerase volume used was 1 &mu;L. The total volume used in each well was 7 &mu;L. R1 and SP6 RNA Polymerase were mixed together and incubated for 15 minutes at 30&deg;C. Next, R4 was added to the mixture and the solution was incubated for another 15 minutes. The composition of each lane is given below:


::{| border = "1" class="wikitable" style="margin:auto;"
! scope="col" | Lane
! scope="col" | Components
|- align="center"
| 1|| 10 base pair ladder
|- align ="center"
| 2|| R1 aptamer
|- align ="center"
| 3|| R4 Strand
|- align ="center"
|4|| R1 aptamer + SP6 RNA Polymerase
|-align ="center"
|5|| R1 aptamer + SP6 RNA Polymerase + R4 aptamer
|}
<h3><font size="4.5">2.4 Unbound Aptamer-Kleptamer Interactions: Gel Electrophoresis</font></h3>
<h3><font size="4.5">2.4 Unbound Aptamer-Kleptamer Interactions: Gel Electrophoresis</font></h3>
For this gel, the concentrations used for R1 aptamer and K1 strands are all 1 &mu;M. The R1 and K1 aptamers were mixed into solution together at the same concentration and incubated at 30°C for 10 minutes. The composition of each lane is given below:
::{| border = "1" class="wikitable" style="margin:auto;"
! scope="col" | Lane
! scope="col" | Components
|- align="center"
| 1|| 10 base pair ladder
|- align ="center"
| 2|| R1 aptamer
|- align ="center"
| 3|| K1 strand (38 base pair)
|- align ="center"
|4|| K1 strand (23 base pair)
|-align ="center"
|5|| R1 aptamer + K1 strand (38 base pair)
|-align ="center"
|6|| R1 aptamer + K1 strand (23 base pair)
|}
<h3><font size="4.5">2.5 Bistable Mechanisms</font></h3>
<h3><font size="4.5">2.5 Bistable Mechanisms</font></h3>
<h3><font size="4.5">2.6 Oscillatory Mechanisms</font></h3>
<h3><font size="4.5">2.6 Oscillatory Mechanisms</font></h3>
[http://openwetware.org/index.php?title=Biomod/2014/UCR/Breaking_RNA/Acknowledgements&action=edit EDIT]
[http://openwetware.org/index.php?title=Biomod/2014/UCR/Breaking_RNA/Acknowledgements&action=edit EDIT]

Revision as of 02:25, 21 October 2014

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Supplementary

1.1 Oscillator Genes and Strands

DNA Oligonucleotides Base Pair Length Sequences
G1 Non-Template 83 5'-TAA TAC GAC TCA CTA TAG GAT GGC AGC GGA GAG TTG CTT GGA ATG CGT TAT AGT CTC TTA GGT GTG TTC GCA CAC CAC TCT CC-3'
G1 Template 83 5'-GGA GAG TGG TGT GCG AAC ACA CCT AAG AGA CTA TAA CGC ATT CCA AGC AAC TCT CCG CTG CCA TCC TAT AGT GAG TCG TAT TA-3'
G2 Non-Template 56 5'-ATT TAG GTG ACA CTA TAG AGG CGA GAA GCA GCA ATG ATA GTG GAA TTG ACT TAC GC-3'
G2 Template 56 5'-GCG TAA GTC AAT TCC ACT ATC ATT GCT GCT TCT CGC CTC TAT AGT GTC ACC TAA AT-3'
G3 Non-Template 52 5'-TTC TAA TAC GAC TCA CTA TAG CGT AAG TCA ATT CCA CTA TCA TTG CTG CTT C-3'
G3 Template 52 5'-GAA GCA GCA ATG ATA GTG GAA TTG ACT TAC GCT ATA GTG AGT CGT ATT AGA A-3'
G4 Non-Template 83 5'-ATT TAG GTG ACA CTA TAG AGG CGA CGT CGC CTC TCA ACG AAC CTT ACG CAA TAT CAG AGA ATC CAC ACA AGC GTG TGG TGA GAG G-3'
G4 Template 83 5'-CCT CTC ACC ACA CGC TTG TGT GGA TTC TCT GAT ATT GCG TAA GGT TCG TTG AGA GGC GAC GTC GCC TCT ATA GTG TCA CCT AAA T-3'
k1 61 5'-TexRd-CGT CGC CTC TCA ACG AAC CTT ACG CAA TAT CAG AGA ATC CAC ACA AGC GTG TGG TGA GAG G-IowaBlack-3'
​

1.2 Bistable Genes and Strands

DNA Oligonucleotides Base Pair Length Sequences
G1 Non-Template 83 5'-TAA TAC GAC TCA CTA TAG GAT GGC AGC GGA GAG TTG CTT GGA ATG CGT TAT AGT CTC TTA GGT GTG TTC GCA CAC CAC TCT CC-3'
G1 Template 83 5'-GGA GAG TGG TGT GCG AAC ACA CCT AAG AGA CTA TAA CGC ATT CCA AGC AAC TCT CCG CTG CCA TCC TAT AGT GAG TCG TAT TA-3'
G2 Non-Template 57 5'-ATT TAG GTG ACA CTA TAG AGG CGA GCG TAA GTC AAT TCC ACT ATC ATT GCT GCA AGC-3'
G2 Template 57 5'-GCT TGC AGC AAT GAT AGT GGA ATT GAC TTA CGC TCG CCT CTA TAG TGT CAC CTA AAT-3'
k1 100 5'-TexRd-CGT CGC CTC TCA ACG AAC CTT ACG CAA TAT CAG AGA ATC CAC ACA AGC GTG TGG TGA GAG G-IowaBlack-3'
Spinach Non-Template 100 5'-ATT TAG GTG ACA CTA TAG AGG ACG CGA CCG AAA TGG TGA AGG ACG GGT CCA GTG CTT CGG CAC TGT TGA GTA GAG TGT GAG CTC CGT AAC TGG TCG CGT C-3'
Spinach Template 100 5'-GAC GCG ACC AGT TAC GGA GCT CAC ACT CTA CTC AAC AGT GCC GAA GCA CTG GAC CCG TCC TTC ACC ATT TCG GTC GCG TCC TCT ATA GTG TCA CCT AAA T-3'
Malachite Green Non-Template 68 5'-ACT ATG ATA ATA CGA CTC ACT ATA GGG AGA GGA TCC CGA CTG GCG AGA GCC AGG TAA CGA ATG GAT CC-3'
Malachite Green Template 68 5'-GGA TCC ATT CGT TAC CTG GCT CTC GCC AGT CGG GAT CCT CTC CCT ATA GTG AGT CGT ATT ATC ATA GT-3'
​

2.1 Enzyme Inhibition RNA Aptamers: Fluorometry

2.2 Bound Aptamer-Kleptamer Interactions: Fluorometry

For the T7 RNA Polymerase reactivation, Malachite Green was employed as the reporter dye. The transcriptional solution contained the following components:

Component Concentration
Malachite Green Dye 16 μM
Malachite Green Gene 100 nM
MgCl2 0.024 M
NTPs 7.5 mM

The solution also contains RNA Polymerase transcription buffer and RNase free water. 1.3 uL of the T7 RNAP enzyme was added to solution along with 0.7 uL of phase to prevent inhibition due to diphosphate waste. The transcription reactions took place at 30°C. For the experimental cuvette, 800 nM of R3 was added as well as 1.2 μM of R2. The total volume used in each cuvette was 60 μL

SP6 RNA Polymerase

2.3 Bound Aptamer-Kleptamer Interactions: Gel Electrophoresis

For the first gel, the concentration of R1 and K1 used are 1 μM and 2 μM and the T7 RNA Polymerase volume used was 1 μL. The total volume in each well was 7 μL. R1 and SP6 RNAP were mixed together and incubated for 20 minutes at 30°C. Next, K1 was added to the mixture and the solution was incubated for another 10 minutes at 30°C. A list of the components in each well is given below:

Lane Components
1 10 base pair ladder
2 R1 aptamer
3 K1 Strand (23 base pair)
4 K1 Strand (38 base pair)
5 R1 aptamer + SP6 RNA Polymerase
6 R1 aptamer +SP6 RNA Polymerase +K1 (23 base pair)
7 R1 aptamer +SP6 RNA Polymerase +K1 (38 base pair)

For the second gel, the concentrations of R1 and R4 used were both 0.7 μL and the SP6 RNA Polymerase volume used was 1 μL. The total volume used in each well was 7 μL. R1 and SP6 RNA Polymerase were mixed together and incubated for 15 minutes at 30°C. Next, R4 was added to the mixture and the solution was incubated for another 15 minutes. The composition of each lane is given below:

Lane Components
1 10 base pair ladder
2 R1 aptamer
3 R4 Strand
4 R1 aptamer + SP6 RNA Polymerase
5 R1 aptamer + SP6 RNA Polymerase + R4 aptamer

2.4 Unbound Aptamer-Kleptamer Interactions: Gel Electrophoresis

For this gel, the concentrations used for R1 aptamer and K1 strands are all 1 μM. The R1 and K1 aptamers were mixed into solution together at the same concentration and incubated at 30°C for 10 minutes. The composition of each lane is given below:

Lane Components
1 10 base pair ladder
2 R1 aptamer
3 K1 strand (38 base pair)
4 K1 strand (23 base pair)
5 R1 aptamer + K1 strand (38 base pair)
6 R1 aptamer + K1 strand (23 base pair)

2.5 Bistable Mechanisms

2.6 Oscillatory Mechanisms

EDIT

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