User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/22

Karmella's BioBrick Cloning

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05/22/15 Ryan - Bra minipreps & digests Cas-tone - minipreps & digests Ryan - Bra minipreps & digests Minipreps Sigma GenElute kit, elute w/ 75 μL elution sln. Digests Check with EcoRI digests - promoter insertion generates a new EcoRI site Note: BraR = 776, pBra = 214 Reagent Volume Expected: 1-8. pBra-BraR_MRV = 2989, 1202 empty vector = 3977 15 μL/lane; 1% agarose; Ladder DNA(plasmid) 2.0 μL 10X buffer 1.5 EcoRI 1.0 dH2O 10.5   15 μL --> 37°C/ ~15 min. Cas-tone - H3_MV11 minipreps & digests Cultures Pick 2 colonies from each ligation plate Make streak library & 5 mL cultures Digests Check with S/HindIII digests - H3 insertion destroys SpeI site Note: Fragment without H3 part = 7162; added H3-1-# size + 7162 to get lengths for longer H/H band H3-1-40 = 129; H3-1-60 = 189; H3-1-80 = 250; H3-1-116 = 373; H3-1-136 = 433 Reagent Volume Expected: 1,2. H3-1-40_MV11 = 2345, 7291 3,4. H3-1-60_MV11 = 2345, 7351 5,6. H3-1-80_MV11 = 2345, 7412 7,8. H3-1-116_MV11 = 2345, 7535 9,10. H3-1-136_MV11 = 2345, 7595 15 μL/lane; 1% agarose; Ladder DNA(plasmid) 2.0 μL 10X buffer 1.5 SpeI 1.0 HindIII 1.0 dH2O 9.5   15 μL --> 37°C/ ~15 min.