Difference between revisions of "Part:BBa K1332011"
| Line 18: | Line 18: | ||
<br> | <br> | ||
<h4>Protocol</h4> | <h4>Protocol</h4> | ||
| − | 1.RNase processing<br> | + | <b>1.RNase processing</b><br> |
<table class="table" border="2"> | <table class="table" border="2"> | ||
<tr><td></td><td>Group1(RNaseA)</td><td>Group2(RNaseR)</td></tr> | <tr><td></td><td>Group1(RNaseA)</td><td>Group2(RNaseR)</td></tr> | ||
| Line 30: | Line 30: | ||
Incubate them at 37 °C for 20 minutes.<br> | Incubate them at 37 °C for 20 minutes.<br> | ||
<br> | <br> | ||
| − | 2.RT-PCR<br> | + | <b>2.RT-PCR</b><br> |
Mix the following reagents.<br> | Mix the following reagents.<br> | ||
<table class="table" border="2"> | <table class="table" border="2"> | ||
| Line 99: | Line 99: | ||
</table> | </table> | ||
<br> | <br> | ||
| − | 3.Electrophoresis | + | <b>3.Electrophoresis</b> |
<br> | <br> | ||
<h4>Result</h4> | <h4>Result</h4> | ||
Revision as of 10:23, 7 October 2014
Histidine tag (8 AA) and RFP semi-permanent generator
This generator is capable of synthesizing a RFP (+histidine tag) polymer. This generator consists of a circularization device (5’ side), histidine tag (8 AA) and RFP (without stop codon) and a circularization device (3’ side). A mRNA is circular, so translation continues semi-permanently. A synthesis of the RFP (+histidine tag) become possible by a simply transformation, but the coloration of RFP is weak.
Existence proof of circular mRNA
Summary of the experiment
The existence of circular mRNA is confirmed by RNase processing. RNA is decomposed by RNaseA (endoribonuclease). Endogenous RNA (GAPDH) is decomposed by RNaseR (exoribonuclease), but circular RNA is not decomposed. Double-stranded DNA from undecomposed RNA can be gained with RT-PCR. So the existence of circular mRNA is confirmed by the observation of the DNA with electrophoresis.
Flow of the experiment
Purpose: proving the existence of circular mRNA
Goal: finding the RNA that is decomposed by endoribonuclease but is not decomposed by exoribonuclease.
Protocol:
1. RNase processing: to find the circular mRNA
2. RT-PCR: to synthesize cDNA and to detect the cDNA synthesized from circular mRNA or endogenous RNA
3. Electrophoresis: to detect the DNA synthesized from the cDNA
Protocol
1.RNase processing
| Group1(RNaseA) | Group2(RNaseR) | |
| RNaseA | 1 µL | |
| RNaseR | 1 µL | |
| buffer | 1 µL | |
| RNA solution | 9 µL | 8 µL |
| total | 10 µL | 10 µL |
Incubate them at 37 °C for 20 minutes.
2.RT-PCR
Mix the following reagents.
| Group1(RNaseA) | Group2(RNaseR) | Group3(non-treated) | |
| RNA solution(after RNase processing) | 8 µL | 8 µL | |
| RNA solution | 6 µL | ||
| pure water | 2 µL | ||
| Oligo(dT)15primer | 1 µL | 1 µL | 1 µL |
| Random primer | 1 µL | 1 µL | 1 µL |
| total | 10 µL | 10 µL | 10 µL |
Incubate them at 70 °C for 5 minutes.
Incubate them at 4 °C for 5 minutes.
Incubate them on ice.
Mix the following reagents.
| Nuclease Free Water | 4.58 µL |
| GoScript™ 5×reaction buffer | 12.2 µL |
| 25mM MgCl2 | 6.1 µL |
| 10mM PCR Nucleotide Mix | 3.05 µL |
| Recombinant RNasin Ribo nuclease Inhibitor | 1.52 µL |
| GoScript™ Reverse | 3.05 µL |
Add 10 µL of it each to group1,2,3.
Synthesize cDNA.
| Annealing | At 25 °C for 5 minutes |
| Elongation | At 42 °C for 60 minutes |
| Inactivation | At 70 °C for 15 minutes |
| At 4 °C for ∞ |
Mix the following reagents.
| Group1 | Group2 | Group3 | Group4 (total RNA without RNase processing and reverse transcription) | |||||
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
| Nuclease Free Water | 10 µL | 10 µL | 10 µL | 10 µL | 10 µL | 10 µL | 10 µL | 10 µL |
| GoTaq 2×mix | 25 µL | 25 µL | 25 µL | 25 µL | 25 µL | 25 µL | 25 µL | 25 µL |
| primer Fw (to detect circular mRNA) | 2.5 µL | 2.5 µL | 2.5 µL | 2.5 µL | ||||
| primer Rv (to detect circular mRNA) | 2.5 µL | 2.5 µL | 2.5 µL | 2.5 µL | ||||
| primer Fw (to detect liner(endogenous) mRNA) | 2.5 µL | 2.5 µL | 2.5 µL | 2.5 µL | ||||
| primer Rv (to detect liner(endogenous) mRNA) | 2.5 µL | 2.5 µL | 2.5 µL | 2.5 µL | ||||
| cDNA solution (Group1) | 10 µL | 10 µL | ||||||
| cDNA solution (Group2) | 10 µL | 10 µL | ||||||
| cDNA solution (Group3) | 10 µL | 10 µL | ||||||
| RNA solution (Group4) | 10 µL | 10 µL | ||||||
Do the PCR assay.
| At 94°C for 3 minutes | |
| At 94°C for 20 seconds | 35 cycles |
| At 58°C for 20 seconds | |
| At 72°C for 50 seconds | |
| At 72°C for 70 seconds | |
| At 4°C for ∞ |
3.Electrophoresis
Result
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1000
Illegal AgeI site found at 1112 - 1000COMPATIBLE WITH RFC[1000]