Team:MIT gateway
From 2010.igem.org
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<div class="bodybaby" id="general">Commented Protocol</div><br> | <div class="bodybaby" id="general">Commented Protocol</div><br> | ||
<b class="bolded" id="miniprep">Design PCR-Primers</b><br> | <b class="bolded" id="miniprep">Design PCR-Primers</b><br> | ||
- | The Gateway® clones have a reading frame which should be kept. Design primers that the PCR product starts with a ATG and ends with a STOP-codon or the last aminoacid (if you want to make a fusion protein). Primer3plus is a powerful tool helping you to pick primers with the right annealing temperature which should be 60°C. Try to avoid self similarity and other things as usual, but because you are very limited in the position of the primers (its start and stop), I only care about annealing temperature and give it a try. Then just add to the primer which binds the start codon the attB1.1-sequence at his 5' End . To the primer which binds the stop codon or the last aminoacid add the attB2.1-sequence at his 5' End . The open reading frame is indicated and you should change the last two NN to code for an aminoacid of your choice. Good luck for the PCR! Because of the long 5' overhang and the restrictions on picking the primers, getting the PCR to work can be tricky.<br> | + | The Gateway® clones have a reading frame which should be kept. Design primers that the PCR product starts with a ATG and ends with a STOP-codon or the last aminoacid (if you want to make a fusion protein). Primer3plus is a powerful tool helping you to pick primers with the right annealing temperature which should be 60°C. Try to avoid self similarity and other things as usual, but because you are very limited in the position of the primers (its start and stop), I only care about annealing temperature and give it a try. Then just add to the primer which binds the start codon the attB1.1-sequence at his 5' End . To the primer which binds the stop codon or the last aminoacid add the attB2.1-sequence at his 5' End . The open reading frame is indicated and you should change the last two NN to code for an aminoacid of your choice. Good luck for the PCR! Because of the long 5' overhang and the restrictions on picking the primers, getting the PCR to work can be tricky.<br><br> |
- | Improved and more efficient att sites used to recombine into pDONR 221:<br> | + | <u>Improved and more efficient att sites used to recombine into pDONR 221:<br></u> |
attB1.1 GGG-GCA-ACT-TTg-tac-aaa-aaa-gtt-gNN<br> | attB1.1 GGG-GCA-ACT-TTg-tac-aaa-aaa-gtt-gNN<br> | ||
attB2.1 GG-GGC-AAC-TTT-GTA-CAA-Caa-agt-tgN<br> | attB2.1 GG-GGC-AAC-TTT-GTA-CAA-Caa-agt-tgN<br> |
Revision as of 21:41, 18 October 2010
bacterial biobrick construction |
The Mammalian team used Gateway cloning to assemble its composite parts.
1 Gateway Cloning 2 Commented Protocol 2.1 Design PCR-Primers 2.2 Gel Purify PCR Product 2.3 Confirm PCR Product 2.4 Measure DNA 2.5 Calculate PCR Product Needed 2.6 Calculate volume of DONRTM needed 2.7 Prepare Gateway Reaction 2.8 Retrieve 5x BP Clonase II 2.9 Add BP-Clonase to Gateway Reaction 2.10 Incubate 2.11 Add ProteinaseK 2.12 Transform Bacteria 2.13 Plate Bacteria 3 Known Issues 4 Citation |
Gateway Cloning Commented Protocol Design PCR-Primers The Gateway® clones have a reading frame which should be kept. Design primers that the PCR product starts with a ATG and ends with a STOP-codon or the last aminoacid (if you want to make a fusion protein). Primer3plus is a powerful tool helping you to pick primers with the right annealing temperature which should be 60°C. Try to avoid self similarity and other things as usual, but because you are very limited in the position of the primers (its start and stop), I only care about annealing temperature and give it a try. Then just add to the primer which binds the start codon the attB1.1-sequence at his 5' End . To the primer which binds the stop codon or the last aminoacid add the attB2.1-sequence at his 5' End . The open reading frame is indicated and you should change the last two NN to code for an aminoacid of your choice. Good luck for the PCR! Because of the long 5' overhang and the restrictions on picking the primers, getting the PCR to work can be tricky. Improved and more efficient att sites used to recombine into pDONR 221: attB1.1 GGG-GCA-ACT-TTg-tac-aaa-aaa-gtt-gNN attB2.1 GG-GGC-AAC-TTT-GTA-CAA-Caa-agt-tgN The original att sites used to recombine into pDONR 221: attB1 GGGG-ACA-AGT-TTg-tac-aaa-aaa-gca-ggc-tNN attB2 GGG-GAC-CAC-TTT-GTA-CAA-Gaa-agc-tgg-gtN The att sites used to recombine into pDONR P4-P1R: attB4 GGGG-ACA-ACT-TTg-tat-aga-aaa-gtt-gNN attB1 GGG-GAC-TGC-TTT-TTT-GTA-Caa-act-tgN The att sites used to recombine into pDONR P2R-P3: attB2 GGGG-ACA-GCT-TTc-ttg-tac-aaa-gtg-gNN attB3 GGG-GAC-AAC-TTT-GTA-TAA-Taa-agt-tgN Gel Purify PCR Product Confirm PCR Product Measure DNA Calculate PCR Product Needed Calculate volume of DONTR(TM) needed Prepare Gateway Reaction Retrieve 5x BP-Clonase II Add BP-Clonase to Gateway Reaction Incubate Add ProteinaseK Transform Bacteria Plate Bacteria Known Issues Citation 1. Untergasser A. “Cloning – Gateway BP-Reaction II” Untergasser's Lab. Summer 2006. (include here the date when you accessed these page). |