User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/17

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07/17/11 ✓ Cultures/minipreps: KAH219-224 (2 each); p65 Biobrick (clone #3, two new preps from streak) ✓ Assemblies: KAH119-224 into pSB31 (for mouse ES cell expression, Manching Ku) Minipreps > Check with E/P digests Reagent Volume Expected: Gel 1 1,2. KAH219 = 2422 3,4. KAH220 = 1345 5,6. KAH221 = 1645 7,8. KAH222 = 2614 9,10. KAH223 = 1537 11,12. KAH224 = 1837 Gel 2 1,2. p65 Biobrick clone #3 = 783 3. p65 mut1 template = 470, 313 15 μL/lane; 1% agarose 15 μL/lane; 1% agarose DNA(plasmid) 2.0 μL 10X buffer 1.5 EcoRI 1.0 PstI 1.0 dH2O 9.5   15 μL --> 37°C/ ~15 min. --> p65 worked! Combine preps 1/2. Order sequencing rxn. tomorrow. Sequencing results (7/18/11) KAH017v2-3, P0001: good KAH017v2-3, P0002: good Assemblies KAH219/pSB31: (1) KAH219/(E/S blunt)/2422 + pSB31/(E blunt dp)/5127 ✓ KAH220/pSB31: (2) KAH220/(E/S blunt)/1345 + " KAH221/pSB31: (3) KAH221/(E/S blunt)/1645 + " KAH222/pSB31: (4) KAH222/(E/S blunt)/2614 + " KAH223/pSB31: (5) KAH223/(E/S blunt)/1537 + " KAH224/pSB31: (6) KAH224/(E/S blunt)/1837 + " > Digests (Fermentas FD) Reagent Volume   30 μL/lane, 1% agarose DNA (plasmid) 25.0 10x buffer 3.0 EcoRI 1.0 SpeI 1.0 dH2O ---   30 μL --> 37°C/ ~30 min. > Measure concentration Sample OD260 260/280 ng/μL 1. KAH219 (E/S) 0.267 2.17 13.4 2. KAH220 (E/S) 0.518 2.01 25.9 3. KAH221 (E/S) 0.493 1.79 24.7 4. KAH222 (E/S) 0.439 1.97 22.0 5. KAH223 (E/S) 0.256 2.19 12.8 6. KAH224 (E/S) 0.465 2.07 23.3 > Blunting (NEB Quick Blunting kit) Reagent Volume DNA (clean digest) up to 19 μL 10x blunting buffer 2.5 1 mM dNTP mix 2.5 Blunt Enzyme mix 1.0 dH2O ---   25 μL --> R.T./ 15 min.; deactivate @ 70°C/ 10 min. --> Use max. volume for all inserts KAH219 [final] = 10.2 ng/μL KAH220 [final] = 19.7 ng/μL KAH221 [final] = 18.8 ng/μL KAH222 [final] = 16.7 ng/μL KAH223 [final] = 9.7 ng/μL KAH224 [final] = 17.7 ng/μL > Ligations Ligation Plate results (lig : neg crtl) 01/23/11 1. KAH160(E/S blunt)/2422, 24 ng + pSB31(E blunt dp)/5127, 25 ng KAH219/pSB31 2:1 (Pick 4) 2. KAH160(E/S blunt)/1345, 13 ng + " KAH220/pSB31 3:1 (Pick 4) 3. KAH160(E/S blunt)/1645, 16 ng + " KAH221/pSB31 4:1 (Pick 4) 4. KAH160(E/S blunt)/2614, 25 ng + " KAH222/pSB31 3:1 (Pick 4) 5. KAH160(E/S blunt)/1537, 15 ng + " KAH223/pSB31 2:1 (Pick 4) 6. KAH160(E/S blunt)/1837, 18 ng + " KAH224/pSB31 5:1 (Pick 4) 7. pSB31(E blunt dp)/ 25 ng     1 2 3 4 5 6 7 Insert DNA 2.5 1.0 1.0 1.5 1.5 1.0 --- Vector DNA 1.0 1.0 1.0 1.0 1.0 1.0 1.0 2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0 5.0 5.0 T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 dH2O 0.5 2.0 2.0 1.5 1.5 2.0 3.0   10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL --> ~30 min/ r.t.; Add to 30 μL DH5α turbo