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Team:MIT gateway
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Revision as of 16:41, 24 October 2010
iGEM 2010
Gateway Cloning |
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 Purification of the PCR-product is needed to get rid of smaller side-products, which remove primer-dimers which can result in false positive colonies. Remember that you want to clone DNA, so the cutting should be made on the weakest UV-light available and as fast as possible. And of course you NEVER make a picture of the gel before. Use the kid for gel-purification available in your lab. Confirm PCR Product You need a PCR product with the attB1.1 and attB2.2 and the DONRTM vector MUST have attP1 and attP2 sites, or it will not work. The amount of plasmids is not soo important as in a multiple Gateway® reaction, because it is more efficient. If you want to optimize you can calculate equimolar amounts of both plasmids as described in the How to measure DNA. Here we use double the amount of DEST-vector, because most of the ones we use are round and about double the size of the ENTRTM clones. Measure DNA The amount of ENTRTM is not so important as in a multiple Gateway® reaction, because it is more efficient. If you want to optimize you can calculate equimolar amounts of both plasmids as described in the multiple Gateway® protocol. Here we use double the amount of DEST-vector, because most of the ones we use are round and about double the size of the ENTRTM clones. Calculate PCR Product Needed ng needed = (length of the PCR product in bp) x 0.0165 Calculate volume of DONTR(TM) needed µl needed = 75 ng needed / (concentration in ng/µl) The DONR-vector should be tested for low background colonies (due to a mutated ccdB-gene) when transferred in DH5alpha-bacteria. Prepare Gateway Reaction PCR-product ( ? ng) pDONRTM-vector (75 ng) add water to a total volume of 4µl Retrieve 5x BP-Clonase II ...From -20°C. It is most efficiently mixed by pipetting up and down, do not vortex. Add BP-Clonase to Gateway Reaction The enzymes looses 50% activity after 15 freeze-thaw cycles. The advantage of BP-ClonaseTMII is that it can be stored at -20 °C because it contains already the buffer. DO NOT LEAVE OUT. Incubate Incubation over-night will enhance the reaction ca. 5-10 fold. This is especially important for PCR products over 5.000 bp. Add ProteinaseK This step will enhance the reaction ca. 100 fold!!!!. This is different to the LR-reactions which are only enhanced 2 fold by adding the proteinase K!!!! Transform Bacteria For electro competent cells use 1-2 µl, for chemical competent all. Plate Bacteria The resulting ENTRTM-vectors are kanamycin resistant. 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). |
