SBB11Ntbk-NikitPatel
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Nikit Patel 11:30, 14 February 2011 (EST)
- Create 100uM stock of oligos: P_sbp_inR and SS50r
- Followed Cloning by PCR Protocol:
- - Used construction files below to setup PCR
- - Did first part of SOEing for P_sbp basic part
- - Thermocycler Program: 2K55
Construction of P_sbp BglBrick basic part sbb1124 PCR ss50f/P_sbp_inR on MG1655 (247 bp, gp = A) PCR P_sbp_inF/ss50r on MG1655 (492 bp, gp = B) --------------------------------------------------- PCR ss50f/ss50r on A+B (710 bp, EcoRI/BamHI) Sub into pBjh1601KC-Bca1144 (EcoRI/BamHI, 3131+910, L) Product is pBjh1601KC-sbb1124 {P_sbp} --------------------------------------------------- P_sbp_inR Reverse removal of EcoRI site in P_sbp CAATAAATTGCAGAAGTCATGTAGGCCTG P_sbp_inF Forward removal of EcoRI site in P_sbp CAGGCCTACATGACTTCTGCAATTTATTG ss50f sbp aaaccGAATTCatgAGATCTgcggtcgttgtgtaggtatccag ss50r sbp tttggGGATCCcaacatcagcttcaataccgttg
Part sbb1104 {P_fadL} PCR ss29f/ss29r on MG1655 gen. (611bp, EcoRI/BamHI) Sub into pBjh1601KC-Bca1144#5 (EcoRI/BamHI, 3131+910, L) Product is pBjh1601KC-sbb1104 {P_fadL} --------------------------------------------------- ss29fForward Cloning of P_fadL aaaccGAATTCatgAGATCTgctttttcagtcagcgccgccag ss29rReverse Cloning of P_fadL tttggGGATCCgataagtgccactgcgactgcgagagc
Nikit Patel 12:35, 16 February 2011 (EST)
- P_sbp Products A and B
- - Ran preparative gel
- - Lanes 2 and 3 from gel confirmed sizes around 250 and 500 bp respectively
- - Bands were cut
- - Performed Zymo Gel Purification on both A + B
- - Setup and run PCR for SOEing using Zymo Gel Purified DNA as template. (Used 2K55 mode for thermocycler)
- P_fadL Products
- - Ran analytical gel (prepped 2uL DNA + 5uL Dye)
- - Band in lane 2 from gel confirmed size around 600 bp
- - Perform Regular Zymo Cleanup
Nikit Patel 18:17, 18 February 2011 (EST)
- P_sbp SOEing PCR products
- - Ran analytical gel
- - Lane 2 from gel confirmed size around 750 bp. (SOEing worked!)
- - Performed Regular Zymo Cleanup