Organelle DNA Library Construction

Organelle DNA Library Construction

I. NEBULIZATION of DNA
     1. 0.5 - 5 ug DNA in TE (10mM/1mM), 25% glycerol, final volume 500 ul
     2. nebulize for 90-100 sec at 5-10 psi (for a medium size of about 1.5kbp)
     3. Precipitate with EtOH/Ammonium Acetate (Sorvall), wash, dry at 65'C
        to remove NH4+.
   Use 0.5 - 2 ug PER LIBRARY  (for 30 - 100 kbp genome size)

II. END REPAIR
        ~1.0 ug fragmented DNA
         1.5 ul dNTP-mix (stock: 1 mM each1))
         2   units Klenow
         2   units T7 DNA polymerase
         1.5 ul Klenow buffer (10x)
        ---------------------------
      in 15 ul final volume.

    Incubate for 30 min at 12'C. Heat for 5 min to 65'C, let cool down.

III. SIZING
     1. Separate on LM agarose gel (1ug/0.5-cm well) and electro-elute 2)
        fractions (0.5 - 1.0 kbp and 1.0 - 3 kbp; ligate separately)
     2. Add glycogen (1 ul of 20mg/ml stock), precipitate by EtOH/AmAc
        (spin down for 30 min). Dissolve pellet in 10 ul TE.

IV. PHOSPHORYLATION
       10 ul sized DNA
         2 ul rATP   (stock : 10 mM)
         2 ul ligase-buffer 10x (similar to kinase buffer)
         5 units kinase
        ---------------
      in 20 ul final volume

   Incubate for 30 min at 37'C.
   Inactivate enzyme at 65'C for 20 min, let cool down.

V. ANALYTIC AGAROSE GEL
Determine DNA concentration and yield on agarose gel. (Assume 50% loss and 
load corresponding amount of nebulized lambda DNA as a standard.

VI. LIGATION INTO VECTOR AND TRANSFORMATION
        335 ng insert DNA (for medium size of 1 kbp)
        500 ng BluescriptII/SmaI, dephosphorylated (molar ratio = 2 insert:1 vector)
         20 ul rATP (stock 10 mM)
         20 units ligase
         20 ul ligase buffer 10x (account for buffer in kinase reaction)
        ------------------------
     in 200 ul final volume (final DNA concentration = 5 ng/ul)

    Incubate o.n. at 14'C.

VII. TRANSFORMATION
        In E.coli XL1. Expect 20 recombinant clones per ng of insert DNA.

1. Device
a.) E.g., BioNeb Cell Disruption System, from Glas-Col Apparatus Company. http://www.glascol.com/homogenizing_two.html
b.) Solution for small budgets
DNA can be nebulized with a device that is used by asthmatics to inhale aerosols. This device is available in medical supply stores for a few dollars. All you have to do is to add a piece of plastic tube to ensure that the liquid does not disperse too much in the nebulization chamber, which would otherwise reduce the amout of liquid you can recuperate. We used an alpha version of the BioNebulizer, kindly provided by its inventor (S.J.Surzycki, Dept. of Biology and the Institute for Molecular and Cellular Biology, Indiana University), as well as models from medical supply stores. Both work fine.

2. Procedure
Nebulize DNA solution, collect solution from chamber bottom, rinse chamber with 200-500 ul H2O. Reduce volume of rinse with speed vac, combine.