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

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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
Hover name
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
Hover name
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.