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Methods and Materials
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(New page: == Making Parts Sca40-Sca43 == For the formation of Sca40-Sca43, five separate composite parts were prepared beforehand. Of these, four were {<tag!}.{b1006} parts - labeled Sca22-Sca25. ...) |
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== Making Parts Sca40-Sca43 == | == Making Parts Sca40-Sca43 == | ||
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In order to assemble AKL41 with other parts, it was necessary first to remove everything south of the phoA stop codon on AKL41. This was accomplished by doing a PCR using ca998 forward oligo and reverse oligo for <phoA> Mea37. The PCR was done on a 25 uL scale, as specified elsewhere (1). The 2K55 PCR machine program was used. Once the PCR was completed, the PCR product was run on a gel to check for correct sizing - which was expected to be around 2850 bp long. Simultaneously, the product was purified: the band was cut from the gel, dissolved in 3 volumes of ADB buffer at 55C for 5-10 minutes. | In order to assemble AKL41 with other parts, it was necessary first to remove everything south of the phoA stop codon on AKL41. This was accomplished by doing a PCR using ca998 forward oligo and reverse oligo for <phoA> Mea37. The PCR was done on a 25 uL scale, as specified elsewhere (1). The 2K55 PCR machine program was used. Once the PCR was completed, the PCR product was run on a gel to check for correct sizing - which was expected to be around 2850 bp long. Simultaneously, the product was purified: the band was cut from the gel, dissolved in 3 volumes of ADB buffer at 55C for 5-10 minutes. | ||
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'''2. Assembling Sca parts with the PCR product''' | '''2. Assembling Sca parts with the PCR product''' | ||
Latest revision as of 04:09, 19 October 2008
Making Parts Sca40-Sca43
For the formation of Sca40-Sca43, five separate composite parts were prepared beforehand. Of these, four were {<tag!}.{b1006} parts - labeled Sca22-Sca25. The labels correspond with the “tag” parts as follows:
- Sca22: {<AP!}.{b1006}
- Sca23: {<HA!}.{b1006}
- Sca24: {<myc!}.{b1006}
- Sca25: {<FLAG!}.{b1006}
All of the parts were in Cam/Kan (antibiotic) assembly vectors. Additionally, a part consisting of {Pbad}{rbs_pelB>}{<phoA!}{<b1006>} was necessary for the Sca parts to join to. This part was labeled AKL41. Both AKL41 and Sca22-Sca25 were the result of composite parts assembly using 1-2-3 Assembly method.
Parts Sca40-Sca43 are composed of the four different {<tag!}.{b1006} parts, each assembled with AKL41 to create final, composite parts consisting of {Pbad}.{rbs_pelB>}.{<phoA>}.{<tag!}.{<b1006>}. Four such goal parts were created, each for one of the following tags: AP, HA, myc, and FLAG.
1. Modifying AKL41
In order to assemble AKL41 with other parts, it was necessary first to remove everything south of the phoA stop codon on AKL41. This was accomplished by doing a PCR using ca998 forward oligo and reverse oligo for <phoA> Mea37. The PCR was done on a 25 uL scale, as specified elsewhere (1). The 2K55 PCR machine program was used. Once the PCR was completed, the PCR product was run on a gel to check for correct sizing - which was expected to be around 2850 bp long. Simultaneously, the product was purified: the band was cut from the gel, dissolved in 3 volumes of ADB buffer at 55C for 5-10 minutes.
2. Assembling Sca parts with the PCR product
Both the PCR product and the Sca parts were digested. The PCR product was digested using 1ul each of EcoRI, BglII, and DpnI. This was done on a 50 uL scale (2). Simultaneously, the 4uL of the Sca parts were combined with 4uL H2O, 1uL NEB2 buffer, and 0.5 uL each of EcoRI and BglII. Both the product and the Sca parts were digested for a period of 1 hour at 37C. Once digestion was completed, the product and the parts were purified using Zymo cleanup method (3). The cleanup from the PCR product was eluted with 10uL H2O, while each of the four Sca parts were eluted with 6uL of H2O. Following purification, ligation of the PCR product and an Sca part was done on a 10uL scale. Each Sca part - 1uL each - was combined with 1uL of the PCR product, 1uL of ligase buffer, 1uL of H2O, and 0.5 uL of ligase into a 0.5mL Eppendorf tube. The ligations were allowed to sit at room temperature for 30 minutes. Following this procedure, transformation was done using the protocol indicated elsewhere (4).
References:
(1.) Section entitled “PCR (25uL)” on the UC Berkeley “Protocols” page.
(2.) Section entitled “Digestion” on the UC Berkeley “Protocols” page.
(3.) Section entitled “Cleanup with Zymo Columns” on the UC Berkeley “Protocols” page.
(4.) Section entitled “Transformation from Ligation Reaction” on the UC Berkeley “Protocols” page.
