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Team:Hannover/Protocols/Cloning/EMP
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<h2>Labwork</h2><p class="text"><ol><li>Amplify the Megaprimer and dilute the purified product 1:10 before using it for the final EMP reaction.<li>Test different weight ratios of Vector:Megaprimer for the best EMP (Table 1).<li>Perform the EMP in a thermocylcer.<li>Purify the product, dephosphorylate and ligate it.<li>Degrade the source vector by DpnI.<li>Transfer the EMP product into competent bacteria.</ol></p> | <h2>Labwork</h2><p class="text"><ol><li>Amplify the Megaprimer and dilute the purified product 1:10 before using it for the final EMP reaction.<li>Test different weight ratios of Vector:Megaprimer for the best EMP (Table 1).<li>Perform the EMP in a thermocylcer.<li>Purify the product, dephosphorylate and ligate it.<li>Degrade the source vector by DpnI.<li>Transfer the EMP product into competent bacteria.</ol></p> | ||
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<h2>Table 1: EMP Mixes</h2> | <h2>Table 1: EMP Mixes</h2> | ||
<p class="text">Test different ratios of insert and vector DNA amounts. We recommend fours mixes with 10 and 20 ng of insert respectively as well as two mixes lacking the F1 oligonucleotide but contain 80 and 160 ng of insert. | <p class="text">Test different ratios of insert and vector DNA amounts. We recommend fours mixes with 10 and 20 ng of insert respectively as well as two mixes lacking the F1 oligonucleotide but contain 80 and 160 ng of insert. | ||
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<table align="line" width="800px" ><tr><td><table> | <table align="line" width="800px" ><tr><td><table> | ||
<tr><td><b>Volume [μl]</b></td><td><b>Compounds of standard PCR</b></td></tr> | <tr><td><b>Volume [μl]</b></td><td><b>Compounds of standard PCR</b></td></tr> | ||
Revision as of 13:27, 15 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 reaction: while the insert is amplified by a standard PCR in the first (megaprimer), the second integrates the places the insert in the final vector backbone already. To try different weight ratios of vector backbone and megaprimer turned out be helpful in prior experiments. Since a correct nucleotide sequence was important here, the F-530 Phusion® High-Fidelity DNA Polymerase (Thermo Scientific) was used.
Labwork
- Amplify the Megaprimer and dilute the purified product 1:10 before using it for the final EMP reaction.
- Test different weight ratios of Vector:Megaprimer for the best EMP (Table 1).
- Perform the EMP in a thermocylcer.
- Purify the product, dephosphorylate and ligate it.
- Degrade the source vector by DpnI.
- Transfer the EMP product into competent bacteria.
Table 1: EMP Mixes
Test different ratios of insert and vector DNA amounts. We recommend fours mixes with 10 and 20 ng of insert respectively as well as two mixes lacking the F1 oligonucleotide but contain 80 and 160 ng of insert.
EMP temperature program
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Table 2: Phosphorylation Mix
Table 3: Ligation Mix
Table 4: DpnI Digest
