Blocking DNA

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Preparation of Genomic DNA for Blocking

1.     Add 10 M NaOH in the DNA sample (0.1-1 ug/ul) to a final concentration of NaOH of 0.4 M.

2.     Place the DNA sample (in microtube) in boiling water for 40 to 45 min.

3.     Cool the DNA sample on ice.  Add equal volume of 3M NaAc (pH 4.6) and two volumes of 100% ETOH, mix the sample well.

4.     Centrifuge for 10 min, rinse the pellet with 70% ETOH, drain the pellet well.

5.     Add certain volume of TE, add 1/10 volume of 3M NaAc (pH 4.6) and two volumes of 100% ETOH, mix the sample well, then repeat step 4.

6.     Add certain volume of TE, add 1/10 volume of 3M NaAc (pH 7) and two volumes of 100% ETOH, mix the sample well.

7.     Centrifuge for 10 min, drain the tube and rinse pellet with 70% ETOH, dry the pellet, dissolve in the final volume of TE.

Notes:

a.     The final volume (concentration) of the DNA sample is very important for genomic in situ hybridization or genomic blocking (differentiation) analysis.  You may test several times (concentrating or diluting the sample) to get the perfect concentration.  For detecting the alien chromosome in a wheat-alien addition line, the wheat blocking DNA should be 50-300 times more than the probe DNA in hybridization mixture.

b.     You can use other techniques to prepare the blocking DNA, such as autoclaving, shearing the DNA using a syringe and a small needle, or sonicating.  The advantages of this techniques include: i) it is easy to control the size of the DNA (size range from 100 bp to 1 kb with 40-45 min boiling).  ii) the DNA sample will be clean because of the repeated precipitation.