IGEM:Cambridge/2008/Notebook/Voltage/BioBrick Manipulation

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They were then all cut with EcoRI and PstI and ligated into pSB4C5, which had been cut with the same enzyme. This was done at a 16:1 ratio of masses as OsmY is about 250bp and pSB4C5 is 4kbp. This then gave a volume ratio of 6.25μL to 0.4, 0.5 and 0.4μL for A,B and C. TOP10 cells were then transformed with the ligation product with highly competent TOP10 for extract C. The cells were then plated onto 35μg/mL chloramphenicol and grown at 37°C and culture C was found to have grown colonies.
They were then all cut with EcoRI and PstI and ligated into pSB4C5, which had been cut with the same enzyme. This was done at a 16:1 ratio of masses as OsmY is about 250bp and pSB4C5 is 4kbp. This then gave a volume ratio of 6.25μL to 0.4, 0.5 and 0.4μL for A,B and C. TOP10 cells were then transformed with the ligation product with highly competent TOP10 for extract C. The cells were then plated onto 35μg/mL chloramphenicol and grown at 37°C and culture C was found to have grown colonies.
Single colonies were then re PCRed using standard BioBrick protocol and also colonies were innoculated into 10ml LB with 35μg/mL chloramphenicol.
Single colonies were then re PCRed using standard BioBrick protocol and also colonies were innoculated into 10ml LB with 35μg/mL chloramphenicol.
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=OsmY and RBS=
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The RBS to be used is B0030 and the PCR product is used. OsmY plasmid is opened with Spe and RBS is cut with Xba and Spe. Ligation between the 2 is performed but founfd to be unsatisfactory, with the OsmY self ligating too readily. RBS is now cut with just Xba as this leaves it being a relatively large piece so more likely to ligate. In addition purificartion can be carried out - any pieces which are re-circularised have no insert. Now the whole is cut with Spe and then ligated, hopefully circularising the plasmid. A further modification is to use decreasing temperature, from 42-35°C, to cause accurate annealing of the sticky ends.
=Kdp=
=Kdp=

Revision as of 08:31, 14 August 2008

OsmY

OsmY was PCRed out of the biobricks which contained it - BBa_J45996 (A) , BBa_J45995 (B) and BBa_J45250 (C). The primers were designed to have the BioBrick prefix and suffix. After PCRing the DNA concentration was determined with the nanodrop, and determined to be 57.8, 47.8 and 58.8 ng/μL respectively. They were then all cut with EcoRI and PstI and ligated into pSB4C5, which had been cut with the same enzyme. This was done at a 16:1 ratio of masses as OsmY is about 250bp and pSB4C5 is 4kbp. This then gave a volume ratio of 6.25μL to 0.4, 0.5 and 0.4μL for A,B and C. TOP10 cells were then transformed with the ligation product with highly competent TOP10 for extract C. The cells were then plated onto 35μg/mL chloramphenicol and grown at 37°C and culture C was found to have grown colonies. Single colonies were then re PCRed using standard BioBrick protocol and also colonies were innoculated into 10ml LB with 35μg/mL chloramphenicol.

OsmY and RBS

The RBS to be used is B0030 and the PCR product is used. OsmY plasmid is opened with Spe and RBS is cut with Xba and Spe. Ligation between the 2 is performed but founfd to be unsatisfactory, with the OsmY self ligating too readily. RBS is now cut with just Xba as this leaves it being a relatively large piece so more likely to ligate. In addition purificartion can be carried out - any pieces which are re-circularised have no insert. Now the whole is cut with Spe and then ligated, hopefully circularising the plasmid. A further modification is to use decreasing temperature, from 42-35°C, to cause accurate annealing of the sticky ends.

Kdp

Kdp was PCRed out of the chromosome of MG1655, in two different tubes. The primers were designed to have the BioBrick prefix and suffix. After PCRing the DNA concentration was determined with the nanodrop, and determined to be 127.5 and 70.5ng/μL. They were both then cut with EcoRI and PstI and ligated into pSB4C5, which had been cut with the same enzyme. As Kdp is 4.4kb and the vector is 4kb approximatley the 1.1:1 ratio of mass was used (Kdp:pSB4C5), giving a volume ratio of 6.25μL to 3μL and 5.1μL. TOP10 cells were then transformed with the ligation product with highly competent TOP10 for extract 1. The cells were then plated onto 35μg/mL chloramphenicol and grown at 37°C and neither culture was found to have grown colonies.

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