Difference between revisions of "Part:BBa K929301"
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The Potsdam Assembly Standard is a modified PLICing (Phosphorothioate-based ligase-independent gene cloning) method (Blanusa ''et al.'' (2010)). For this standard we designed a new cloning vector with an RFP expression cassette as insert and two new restriction enzyme recognition sites in the suffix and prefix in pSB1C3. In the prefix we added the Apa I recognition site and in the suffix the Sph I recognition site. Both enzymes causing a 3’ overhang with 4 nucleotides. For the cloning process we cut the vector with Apa I and Sph I. In this assembly standard that’s the only step where we have to use restriction enzymes.<br> | The Potsdam Assembly Standard is a modified PLICing (Phosphorothioate-based ligase-independent gene cloning) method (Blanusa ''et al.'' (2010)). For this standard we designed a new cloning vector with an RFP expression cassette as insert and two new restriction enzyme recognition sites in the suffix and prefix in pSB1C3. In the prefix we added the Apa I recognition site and in the suffix the Sph I recognition site. Both enzymes causing a 3’ overhang with 4 nucleotides. For the cloning process we cut the vector with Apa I and Sph I. In this assembly standard that’s the only step where we have to use restriction enzymes.<br> | ||
The insert was amplified with primers that contain 4 phosphothioate nucleotides and the recognition sites for Apa I and Sph I at the 5’ end. After incubation in an iodine/ethanol solution the thiophosphates were cut out resulting in a 3’ overhang which is suitable to the overhangs that was created by cutting the new assembly vector. | The insert was amplified with primers that contain 4 phosphothioate nucleotides and the recognition sites for Apa I and Sph I at the 5’ end. After incubation in an iodine/ethanol solution the thiophosphates were cut out resulting in a 3’ overhang which is suitable to the overhangs that was created by cutting the new assembly vector. | ||
After that the digested vector and the PLICed insert were mixed and transformed into <i>E. coli</i>. By using the RFP expression cassette we created a ligation control system. Due to the fact that red fluorescent colonies have a failed vector ligation we can tell which colonies are correctly ligated. The colonies that do not show red fluorescence are the positive clones.<br><br> | After that the digested vector and the PLICed insert were mixed and transformed into <i>E. coli</i>. By using the RFP expression cassette we created a ligation control system. Due to the fact that red fluorescent colonies have a failed vector ligation we can tell which colonies are correctly ligated. The colonies that do not show red fluorescence are the positive clones.<br><br> | ||
Revision as of 14:29, 24 September 2012
Potsdam Standard Backbone
The Potsdam Assembly Standard is a modified PLICing (Phosphorothioate-based ligase-independent gene cloning) method (Blanusa et al. (2010)). For this standard we designed a new cloning vector with an RFP expression cassette as insert and two new restriction enzyme recognition sites in the suffix and prefix in pSB1C3. In the prefix we added the Apa I recognition site and in the suffix the Sph I recognition site. Both enzymes causing a 3’ overhang with 4 nucleotides. For the cloning process we cut the vector with Apa I and Sph I. In this assembly standard that’s the only step where we have to use restriction enzymes.
The insert was amplified with primers that contain 4 phosphothioate nucleotides and the recognition sites for Apa I and Sph I at the 5’ end. After incubation in an iodine/ethanol solution the thiophosphates were cut out resulting in a 3’ overhang which is suitable to the overhangs that was created by cutting the new assembly vector.
After that the digested vector and the PLICed insert were mixed and transformed into E. coli. By using the RFP expression cassette we created a ligation control system. Due to the fact that red fluorescent colonies have a failed vector ligation we can tell which colonies are correctly ligated. The colonies that do not show red fluorescence are the positive clones.