how to isolation pTARBAC2.1 BAC clones from DH10b ？I have tried several times follow many protocol，but still failed. the concentration is higher than expected，but gel electro doesn't appearance the band of BAC.
and i wonder the pTARBAC2.1 is high or low copy in DH10B.
Which method are you following? and Please explain your question briefly.
Culture volume 100 ml
resuspend pellet in 8 ml of 50 mM Tris/HC, 10 mM EDTA, RNaseA 100µg/ml
add 8 ml 200 mM NaOH, 1% SDS; mix the suspension gently by inverting the tube several times until a homogenous suspension is formed. Incubate at RT for 5 min not longer.
add 8 ml of 2.8 M KAc, pH5.1, mix the suspension gently by invertin the tube several times . Keep on ice 15 min.
Centrifuge at 12 000g, at 4°; 15 min. Carefully remove the supernatant in a 50 ml plastic tube.
Use cartridge column (like Nucleobond AX100) / or Ph-OH:CCl4 extraction.
Precipitate the DNA with isopropanol.
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