A query

Nasser Mahna n_mahna at yahoo.com
Fri Jan 27 06:13:10 EST 2006

I would like to ask about a problem I have faced. I am trying to do a double digest, which I have done it many times before, to isolate an insert from the pBlueScript vector. But I don’t know because of what reason some thing is degrading the DNA of the vector. To be sure what is happening there in the reaction mixtures and what is indeed wrong with them, I tried to verify each component of the reaction mixture, which probably is the cause of this fail. I put the following reactions:
  1) DNA + water ; 2) New digestion buffer of Promega (MultiCore) + water + DNA; 3) An older buffer of MutiCore + water + DNA; 4) RE reaction without BSA + BamHI; 5) RE reaction without BSA + KpnI; 6) Complete RE reaction mixture + BamHI; 7) Complete RE reaction mixture + KpnI; 8) Complete RE reaction mixture +  BamHI + KpnI; I incubated all mixtures in a PCR machine at 37°C for one hour without putting the lid because of its high temperatue. Finally I deactivated the RE reaction at 75 °C for 10 min. In addition, I put a control for the DNA.
  The result was nothing in the gel after electrophoresis but for “DNA + water” and “Control“.
  I would appreciate if you help me to find out what is the reason for this bad luck.

Nasser Mahna
Lab for Fruit Breeding and Biotechnology 
Willem de Croylaan 42, B-3001 Heverlee,
Tel: +32-16-322413
Mobile: +32-477352089
Fax: +32-16-322966
E-mail: nasser.mahna at student.kuleuven.be
           n_mahna at yahoo.com

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