User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/11

Karmella's BioBrick Cloning

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05/11/15 Cancer PcTF - cultures for KAH87_MV10 clone #1 from streak library (clone #3 is wrong) Cas-tone - PCR-amplify CMV Ryan - minipreps & digests Cas-tone Project STAGE 1 - pcDNA-dCas9-VP64 vector re-design Knock-out VP64 from C-terminus - replace AscI-VP64:HA:STOP-EcoRI-XbaI with AscI-HA:STOP-XbaI dsOligo 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 PCR-clone histone parts as XbaI-Kozak-histone part-a-NotI. Note: Do no use Kozak BioBrick, since this will introduce unnatural amino acids onto conserved histone N-terminal tail Order primers STAGE 3 - Cas-fusion Design primers for H2A, H2B, H3, H4, and tails Insert Kozak-histone parts (X/N) into dCas9 (S/N) STAGE 4 - gRNA Put pre-existing gRNA (from luc experiment) into pSPgRNA Application Cas and gRNA plasmids will be co-transformed Analyze Cas protein via Western blot Knock-out FLAG from N-terminus PCR-amplify promoters 2x 50 μL rxns Template = CMV (KAH f primer: XbaI-CMV f1 - 5'-CCTTTCTAGAGTTGACATTGATTATTGGCTAG r primer: SacII-NS-CMV r1 - 5'-CATTCCGCGGGCGGCCGCTACTAGTGAGCTCTGC Reagent Vol Mix (x2) Expected: 1. CMV = 588 5 μL/lane; 1% agarose; Ladder plasmid DNA 0.5 1.0 10 μM XbaI-CMV f1 1.0 2.0 10 SacII-NS-CMV r1 1.0 2.0 2x GoTaq green 25.0 50.0 dH2O 22.5 45.0   50.0 μL --- Thermal cycler: Labnet - GOTAQ 95°C 3 min. 30x[95°C, 30 sec; 55°C 30 sec; 72°C 30 sec] 72°C 3 min. 4°C, ∞ Conclusions Success! Proceed to PCR purification (PCR Clip & Clone protocol) PCR clean-up Qiagen PCR Purification kit Elute & back-elute w/ 30 μL elution buffer Measure [DNA] Sample OD 260 260/280 ng/μL 1. XbaI-CMV-SpeI-NotI-SacII PCR 0.186 1.868 186.4 Minipreps Use Sigma GenElute; elute with 75 μL elution solution Cut w/ E/KpnI See my version of MRV map: https://benchling.com/s/oJ73uatK/edit Note: BbsI drop-in of promoter knocks out KpnI site (54); no E, X, or S sites are restored by promoter insert Note: EcoRI (1025) and XbaI (1040) were maintained after Regulator insertion pAub-AubR/MRV pBja-BjaR/MRV pBra-BraR/MRV pRpa-RpaR/MRV Reagent Volume Expected: 1,2. pAub/AubR/MRV = 4240 3,4. pBja/BjaR/MRV = 4099 5,6. pBra/BraR/MRV = 4191 7,8. pRpa/RpaR/MRV = 4116 9. Empty MRV = 2230, 971 np pro. Reg/MRV = ~4000, 971 15 μL/lane; 1% agarose; Ladder DNA(plasmid) 2.0 μL 10X buffer 1.5 EcoRI 1.0 KpnI 1.0 dH2O 9.5   15 μL --> 37°C/ ~10 min. Measure [DNA] Sample OD 260 260/280 ng/μL 1. pAub-AubR/MRV-1 0.152 1.930 152.3 2. pAub-AubR/MRV-2 0.079 1.992 78.6 3. pBja-BjaR/MRV-1 0.093 1.989 92.7 4. pBja-BjaR/MRV-2 0.043 1.934 43.1 5. pBra-BraR/MRV-1 0.070 2.021 69.9 6. pBra-BraR/MRV-2 0.030 1.946 29.8 7. pRpa-RpaR/MRV-1 0.093 1.956 92.6 8. pRpa-RpaR/MRV-2 0.033 2.000 32.7 Conclusions Clones 1, 7, 8 look like empty Regulator-MRV vectors