Team:MIT gateway

From 2010.igem.org

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<u>Improved and more efficient att sites used to recombine into pDONR 221:<br></u>
<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><br>
-
The original att sites used to recombine into pDONR 221:<br>
+
<u>The original att sites used to recombine into pDONR 221:<br></u>
attB1  GGGG-ACA-AGT-TTg-tac-aaa-aaa-gca-ggc-tNN<br>
attB1  GGGG-ACA-AGT-TTg-tac-aaa-aaa-gca-ggc-tNN<br>
attB2  GGG-GAC-CAC-TTT-GTA-CAA-Gaa-agc-tgg-gtN<br>
attB2  GGG-GAC-CAC-TTT-GTA-CAA-Gaa-agc-tgg-gtN<br>
-
The att sites used to recombine into pDONR P4-P1R:<br>
+
<u>The att sites used to recombine into pDONR P4-P1R:<br></u>
attB4  GGGG-ACA-ACT-TTg-tat-aga-aaa-gtt-gNN<br>
attB4  GGGG-ACA-ACT-TTg-tat-aga-aaa-gtt-gNN<br>
-
attB1  GGG-GAC-TGC-TTT-TTT-GTA-Caa-act-tgN<br>
+
attB1  GGG-GAC-TGC-TTT-TTT-GTA-Caa-act-tgN<br><br>
-
The att sites used to recombine into pDONR P2R-P3:<br>
+
<u>The att sites used to recombine into pDONR P2R-P3:<br></u>
attB2  GGGG-ACA-GCT-TTc-ttg-tac-aaa-gtg-gNN<br>
attB2  GGGG-ACA-GCT-TTc-ttg-tac-aaa-gtg-gNN<br>
-
attB3  GGG-GAC-AAC-TTT-GTA-TAA-Taa-agt-tgN <br>
+
attB3  GGG-GAC-AAC-TTT-GTA-TAA-Taa-agt-tgN <br><br>
<b class="bolded" id="miniprep">Gel Purify PCR Product</b><br>
<b class="bolded" id="miniprep">Gel Purify PCR Product</b><br>

Revision as of 21:42, 18 October 2010



bacterial biobrick construction

The Mammalian team used Gateway cloning to assemble its composite parts.


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). .