User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/23

Karmella's BioBrick Cloning

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04/23/15 Ryan - Receiver cloning stage 2 Rene - sequencing for g053-2, g054 Ryan - Receiver plasmids, assembly Stage 1 - insert Regulator ORFS Phusion PCR-amplify ORFs (add 5'-E, 3'-X cut sites) Insert ORF(E/X) into Vector (E/X). see Vector plasmid map Stage 2 - insert Promoters Cut & dephos promoters with E/X Insert promoters(E/X) into Vector/Regulator (BbsI) via Lig/Dig cycling Assemblies pAub/AubR/MRV: pAub(E/Xdp)/153 + AubR/MRV(BbsI)/4087 pBja/BjaR/MRV: pBja(E/Xdp)/122 + BjaR/MRV(BbsI)/3977 pBra/BraR/MRV: pBra(E/Xdp)/214 + BraR/MRV(BbsI)/3977 pRpa/RpaR/MRV: pRpa(E/Xdp)/136 + RpaR/MRV(BbsI)/3980 Measure conc.'s Sample OD260 260/280 ng/μL 1. AubR/MRV 0.258 1.906 257.7 2. BjaR/MRV 0.177 1.904 177.3 3. RpaR/MRV 0.246 1.896 246.2 Digests (Fermentas FD) E/X Inserts are gBlocks = 2.0 ng/μL Reagent Volume DNA (20 ng) 10.0 μL 10x buffer 2.0 EcoRI 1.0 XbaI 1.0 dH2O 6.0   20.0 μL --> 37°C/ ~15 min. Dephosphorylation (Roche) Assuming that shrimp alkaline phosphatase works in FastDigest buffer conditions Link Reagent Volume DNA (digest) 20.0 μL (20 ng) phosphatase 1.0 dH2O ---   20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~1 ng/μL Digestion/Ligation Reactions (borrowed from Rene's gRNA/Cas9 assembly procedure) Inserts are gBlocks = ~2ng/μL For an insert = ~200 and vector = ~4000, need 5 ng insert for 2:1 insert:vector(50 ng) ratio pAub/AubR/MRV: pAub(E/Xdp)/153 + AubR/MRV(BbsI)/4087 pBja/BjaR/MRV: pBja(E/Xdp)/122 + BjaR/MRV(BbsI)/3977 pRpa/RpaR/MRV: pRpa(E/Xdp)/136 + RpaR/MRV(BbsI)/3980 Note: BraR cloning was not successful, omit from this round of assembly   1 2 3 Insert DNA 5.0 5.0 5.0 Vector DNA 0.2 0.3 0.2 10x FD buf 2.0 2.0 2.0 10 mM DTT 1.0 1.0 1.0 10 mM ATP 1.0 1.0 1.0 FD BbsI/BpiI 1.0 1.0 1.0 T4 ligase (Roche) 1.0 1.0 1.0 dH2O 8.8 8.7 8.8   20.0 μL 20.0 μL 20.0 μL Thermal cycler: Labnet - BbsI Dig/Lig 6x[37°C, 5 min.; 23°C 5 min.] 4°C, ∞ Rene - sequencing g53-2, f (P139) - Confirmed g53-2, r (P140) - low Phred 20 score g54, f (P139) - Confirmed g54, r (P140) - low Phred 20 score