User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/26

Karmella's BioBrick Cloning

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05/26/15 Ben - luc replacement donor plasmid DBN007 Rene - CRISPR PCR library PCR trial Ben - luc replacement donor plasmid Assembly: DBN007_pSB1A3: DBN006(BsaI)/1972 + DBN001(BsaI)/1267+1069 (=2336) Phusion PCR PCR-amplify HPK-CFP gene (DBN006) with new/ improved primers DBN006: https://benchling.com/s/XoQEXLOz/edit Primers: P072, P073 Use Life Tech/ Thermo Tm calculator to calculate annealing temperature Reagent Rxn1,2 Expected: 1,2. DBN006 = 1972 5 μL/lane, 1% agarose; Ladder Template 0.2 10 uM fwd primer 1.0 10 uM rev primer 1.0 10 mM dNTPs 1.0 Phusion pol. 0.5 5x HF buffer 5.0 dH2O 41.3   50.0 Bio-Rad: "Phusion" (block #) 98°C, 3 min 30x[98°C, 10 sec; 67°C, 30 sec; 72°C, 30 sec] 72°C, 10 min 4°C ∞ CONCLUSION Non-specific amplification. Need to gel purify. Use 30 uL of each per lane for gel purification. CONTINUED: 5/27/15 Gel purification Use Sigma kit Elute & back-elute w/ 25 μL elution sln. Sample OD260 260/280 ng/μL 1. DBN006 PCR 0.002 -19 2.0 Concentration is weird. Use max amt. of DNA for subsequent steps Digest & Dephosphorylate the Insert(s) Use BsaI Reagent Volume DNA (500 ng) up to 16.0 μL 10X FD buffer 2.0 FD BsaI 1.0 Roche SAP 1.0 dH2O x μL   20.0 Thermal cycler program: Note: In the Haynes lab, use the LabNet OptiMax Thermocycler, Program "AnOlig RD" 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, ∞ The final concentration is ??? Ligations DBN001 2-part backbone from 05/15/15 2:1 ratio calc.: 1972 bp insert / 2336 bp vector * 2 * 50 = 84 ng insert DBN006/(BsaI)/1972 + DBN001/(BsaI)/2336 DBN001/(BsaI)/2336 Reagent Rxn1 Rxn2 Insert DNA 6.0 --- Vector DNA 3.0 3.0 2x lgn buf (Roche) 10.0 10.0 T4 ligase (NEB) 1.0 1.0 dH2O --- 6.0   20.0 μL 20.0 μL Incubate at RT/ 10 min. Add to 50 μL DH5α-turbo; ice/ 5min. Plate on 100 μg/mL amp agar RESULTS (5/28/15) Success! 50 colonies on ligation plate, 5 colonies on neg. ctrl. Pick two colonies Rene - CRISPR PCR library PCR trial Part 1 - GoTaq "Dirty" PCR on liquid mini-cultures, 600 bp amplicons; primers P163/P215 Part 2 - Nested SYBR real time PCR & melt curves on (1) mini cultures, (2) amplicons from GoTaq Part 1 - GoTaq "Dirty" PCR on liquid mini-cultures, 600 bp amplicons Clone labeling notes: Lu34 = Luc14 cells treated with gRNA034 Ga34 = Gal4EED/luc cells treated with gRNA034 A/B = 96-well plate A or B A01, A02, etc. = well position in dish or spot on agar array 1-12. Luc14 g034 - Lu34_AA01 - Lu34_AA12 13-24. Gal4EED g034 Ga34_AA1 - GaA_12 Reagent Rxn1-24 Mix (x25) Expected: 1-14. PCR insert/ pJET = ~600 bp 10 μL/lane, 1% agarose; Ladder Template (culture) 0.5 --- 10 uM fwd primer 1.0 25.0 10 uM rev primer 1.0 25.0 2x GoTaq (clear) 12.5 312.5 dH2O 10.0 250.0   25.0 μL Labnet: "GoTaq" 98°C, 3 min 35x[95°C, 10 sec; 60°C, 10 sec; 72°C, 20 sec] 72°C, 3 min 4°C ∞ CONCLUSIONS Success with dirty PCR Continue with remainder of dirty PCR & possible SYBR PCR tomorrow