User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/21

Karmella's BioBrick Cloning

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05/21/15 Cas-tone - Assemblies, H3's + MV11 Ryan - pick pBra-BraR-MRV colonies Cas-tone Project STAGE 1 - pcDNA-dCas9-VP64 vector re-design DONE - Knock-out VP64 from C-terminus - replace AscI-VP64:HA:STOP-EcoRI-XbaI with AscI-HA:STOP-XbaI dsOligo DONE - Knock-out FLAG from N-terminus - replace SpeI-CMV:3xFLAG:NLS-SacII with PCR-amplified (SpeI)/XbaI-CMV-SpeI-NotI-SacII; use reverse primer to add SacII STAGE 2 - Histone parts DONE - Order primers (annotated in Benchling) DONE PCR-amplify histone parts as XbaI-Kozak-histone part-a-NotI. Note: Do no use Kozak BioBrick, since this will add extra amino acids onto conserved histone N-terminal tail. Instead, use the Kozak-like seq. from the Addgene plasmid, +3 extra Kozak bases (included in primer) STAGE 3 - Cas-fusion Insert Kozak-histone parts (X/N) into dCas9 plasmid "MV11" (S/N) STAGE 4 - gRNA Put pre-existing gRNA (from luc experiment) into pSPgRNA Assemblies H3-1-40_MV11: H3-1-40/(X/N)/129 + MV11/(S/N)/9506 H3-1-60_MV11: H3-1-60/(X/N)/189 + " H3-1-80_MV11: H3-1-80/(X/N)/250 + " H3-1-116_MV11: H3-1-116/(X/N)/373 + " H3-1-136_MV11: H3-1-136/(X/N)/433 + " Digest of vector (Fermentas FD) SpeI/NotI Reagent Volume DNA (plasmid) 15.0 10x buffer 3.0 SpeI 1.0 NotI 1.0 dH2O 10.0   30 μL --> 37°C/ ~15 min. Gel purification Sigma GenElute kit. Elute & back elute w/ 25 μL elution sln. DpnI digest & clean-up of PCR products Add 1 μL DpnI to each PCR rxn. Incubate @ 37°C/ 5 min. Use the Qiagen Qiaquick PCR Purification kit (cat. no. 28104 ) to purify DNA. Elute & back-elute with 30 μL of elution solution. Measure [DNA] for vector and inserts Sample OD260 260/280 ng/μL 1. MV11 (S/N) 0.03 1.879 13.5 2. H3-1-40 PCR 0.03 1.879 29.8 3. H3-1-60 PCR 0.04 1.974 39.7 4. H3-1-80 PCR 0.047 1.865 46.8 5. H3-1-116 PCR 0.06 1.887 59.57 6. H3-1-136 PCR 0.062 1.932 62.0 Digest & dephos inserts Reagent Volume DNA (500 ng) up to 15.0 μL 10X FD buffer 2.0 FD EcoRI 1.0 FD XbaI 1.0 Roche SAP 1.0 dH2O x μL   20.0 Thermal cycler: LabNet OptiMax "AnOlig shrt" 37°C, 10 min 95°C, 5 min Ramp down to 25°C, 5°C/1 min. [90/1 min, 85/1 min, 80/1 min, ... 25°C/1 min] 25°C, ∞ Final concentration = 25 ng/μL. Ligations > 2:1 ratio calculations... 433 bp largest insert / 9506 bp vector * 2 * 50 ng vector = 4.5 ng 129 bp smallest insert / 9506 bp vector * 2 * 50 ng vector = 1.4 ng Reagent Rxn1-5 Rxn6 Insert DNA 0.5 --- Vector DNA 3.5 3.5 2x lgn buf (Roche) 5.0 5.0 T4 ligase (NEB) 1.0 1.0 dH2O --- 0.5   10.0 μL 10.0 μL RESULTS (5/22/15) Success! Ligation plates have 15-20 colonies, neg. ctrl has 1 colony Pick 2 colonies from each plate for 5 mL cultures and streaks Ryan - pick pBra-BraR-MRV colonies" pBra-BraR-MRV is the only receiver with no potential successes so far Pick 8 colonies form the traditional ligation plate for 5 mL cultures (o/n) and streaks