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Team:Hannover/Protocols/Cloning/EMP
From 2014.igem.org
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| - | <h1><a href="https://2014.igem.org/Team:Hannover/Protocols | + | <h1><a href="https://2014.igem.org/Team:Hannover/Protocols">Protocols</a> / Exponential Megaprimer PCR (EMP)</h1> |
| - | <p class="text">The exponential megaprimer PCR (EMP) was used to replace the metal-binding-related sequences of our pORE-E3_2x35S_T4MBP by GFP. In theory, the EMP is based on two PCR | + | <p class="text">The exponential megaprimer PCR (EMP) was used to replace the metal-binding-related sequences of our pORE-E3_2x35S_T4MBP by GFP. In theory, the EMP is based on two PCR reactions: The insert is amplified in the first (EMP1) and a 3′ overhang is added by a standard PCR. The product of this EMP1 ist called megaprimer. The second places the insert in the final vector backbone already (EMP2). To screen different amounts of megaprimer with and without using the second reverse primer (R2) turned out be helpful in prior experiments (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053360">Jung et. al., 2012</a>). Since a correct nucleotide sequence was important here, the F-530 Phusion® High-Fidelity DNA Polymerase (Thermo Scientific) was used.</p> |
| - | <h2>Workflow</h2><p class="text"><ol><li>Isolate both the source and final vector.<li>Use a <a href="https://2014.igem.org/Team:Hannover/Protocols/Cloning/PCR">standard PCR</a> to add single-stranded 3´end overhangs to the megaprimer and | + | <h2>Workflow</h2><p class="text"><ol><li>Isolate both the source and final vector.<li>Use a <a href="https://2014.igem.org/Team:Hannover/Protocols/Cloning/PCR">standard PCR</a> to add single-stranded 3´end overhangs to the megaprimer and purify the product before using it for the final EMP reaction.<li>Test different weight ratios of Vector:Megaprimer for the best EMP (Table 1).<ul><li>Test different ratios of insert and vector DNA amounts. We recommend to <u>test five mixes</u> including: 10 ng of megaprimer, 20 ng of megaprimer, two mixes lacking the F1 oligonucleotide but containing 80 and 160 ng of megaprimer respectively and a last one containing F1 as well as 160 ng of megaprimer.</li></ul><li>Modify the EMP2 product.<ul><li>Phosphorylate its 5´ends (Table 2)</li><li>Circularize the EMP2 product by adding 1 µl ATP and 1 µl T4 DNA Ligase. Incubate the mix at 16 °C overnight and inactivate the enzyme for 10 min at 80 °C.</li><li>Degrade the source vector by adding 1 µl of DpnI. Incubate the mix for 15 min at 37 °C and inactivate the enzyme for 15 min at 80 °C.</li></ul><li>Transfer the EMP product into <a href="https://2014.igem.org/Team:Hannover/Protocols/Transformation/E.coli_Heatshock">competent bacteria</a>.</ol></p> |
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<h2>Table 1: EMP Mixes and Temperature Program</h2> | <h2>Table 1: EMP Mixes and Temperature Program</h2> | ||
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<table align="line" width="800px" valign="top"><tr><td><table> | <table align="line" width="800px" valign="top"><tr><td><table> | ||
| - | <tr><td><b>Volume [μl]</b></td><td><b>Compounds of | + | <tr><td><b>Volume [μl]</b></td><td><b>Compounds of standard PCR</b></td></tr> |
<tr><td>4.00</td><td>5 x Phusion® HF buffer</td></tr> | <tr><td>4.00</td><td>5 x Phusion® HF buffer</td></tr> | ||
<tr><td>1.00</td><td>10 mM dNTPs</td></tr> | <tr><td>1.00</td><td>10 mM dNTPs</td></tr> | ||
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<tr><td> ~</td><td>megaprimer</td></tr> | <tr><td> ~</td><td>megaprimer</td></tr> | ||
<tr><td>0.20</td><td>Phusion® High-Fidelity DNA Polymerase</td></tr> | <tr><td>0.20</td><td>Phusion® High-Fidelity DNA Polymerase</td></tr> | ||
| - | <tr><td> ~</td><td>20-50 ng template</td></tr><tr><td colspan="2">ad 20 µl | + | <tr><td> ~</td><td>20-50 ng template</td></tr><tr><td colspan="2">ad 20 µl H<sub>2</sub>O</td></tr></table></td><td> |
| - | <table colspan="2" ><tr><td><b> | + | <table colspan="2" ><tr><td><b>Cycler Program</b></td></tr><tr><td>Step</td><td>Temperature [°C]</td><td>Time [min]</td><td>Cycle no.</td></tr><tr><td>1.</td><td>98</td><td>2.0</td><td>1</td></tr><tr><td>2.</td><td>98</td><td>0.5</td><td>35</td></tr><tr><td>3.</td><td>T<sub>A</sub></td><td>0.5</td><td>35</td></tr> |
<tr><td>4.</td><td>72</td><td>-</td><td>35</td></tr></table></td></tr></table></td></tr></table> | <tr><td>4.</td><td>72</td><td>-</td><td>35</td></tr></table></td></tr></table></td></tr></table> | ||
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<tr><td>15.0 µl</td><td>EMP product</td></tr> | <tr><td>15.0 µl</td><td>EMP product</td></tr> | ||
<tr><td>2.0 µl</td><td>10 x Ligase buffer </td></tr> | <tr><td>2.0 µl</td><td>10 x Ligase buffer </td></tr> | ||
| - | <tr><td>2.0 µl</td><td> | + | <tr><td>2.0 µl</td><td>10 mM ATP</td></tr> |
| - | <tr><td>1.0 µl</td><td>Polynucleotide kinase</td></tr></table></td><td><table colspan="2"><tr><td><b> | + | <tr><td>1.0 µl</td><td>Polynucleotide kinase</td></tr></table></td><td><table colspan="2"><tr><td><b>Cycler Program</b></td></tr><tr><td>Step</td><td>Temperature [°C]</td><td>Time [min]</td><td>Cycle no.</td></tr><tr><td>1.</td><td>37</td><td>10</td><td>1</td></tr><tr><td>2.</td><td>80</td><td>10</td><td>1</td></tr></table></td></tr></table> |
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Latest revision as of 23:33, 17 October 2014
Protocols / Exponential Megaprimer PCR (EMP)
The exponential megaprimer PCR (EMP) was used to replace the metal-binding-related sequences of our pORE-E3_2x35S_T4MBP by GFP. In theory, the EMP is based on two PCR reactions: The insert is amplified in the first (EMP1) and a 3′ overhang is added by a standard PCR. The product of this EMP1 ist called megaprimer. The second places the insert in the final vector backbone already (EMP2). To screen different amounts of megaprimer with and without using the second reverse primer (R2) turned out be helpful in prior experiments (Jung et. al., 2012). Since a correct nucleotide sequence was important here, the F-530 Phusion® High-Fidelity DNA Polymerase (Thermo Scientific) was used.
Workflow
- Isolate both the source and final vector.
- Use a standard PCR to add single-stranded 3´end overhangs to the megaprimer and purify the product before using it for the final EMP reaction.
- Test different weight ratios of Vector:Megaprimer for the best EMP (Table 1).
- Test different ratios of insert and vector DNA amounts. We recommend to test five mixes including: 10 ng of megaprimer, 20 ng of megaprimer, two mixes lacking the F1 oligonucleotide but containing 80 and 160 ng of megaprimer respectively and a last one containing F1 as well as 160 ng of megaprimer.
- Modify the EMP2 product.
- Phosphorylate its 5´ends (Table 2)
- Circularize the EMP2 product by adding 1 µl ATP and 1 µl T4 DNA Ligase. Incubate the mix at 16 °C overnight and inactivate the enzyme for 10 min at 80 °C.
- Degrade the source vector by adding 1 µl of DpnI. Incubate the mix for 15 min at 37 °C and inactivate the enzyme for 15 min at 80 °C.
- Transfer the EMP product into competent bacteria.
Table 1: EMP Mixes and Temperature Program
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Table 2: Phosphorylation Mix and Temperature Program
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