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moromou @ mitbbs.com

Use linearized plasmid DNA(gel purified) as template, in vitro transcribe RNA probe, treat with DNase, run gel.

Annoyingly, I got two bands, one is the right size, about 600bp, the other one is about 1.2kb?

I don't think it's the template, because linearized template is about 4.6kb, circular template plasmid is around 3.5kb.

so what's the other band???????

I synthesized 8 probes, two of them are good, one right size band, the linearized templates are borrowed from a labmate.

My probes are cloned in pCR2.1 vector, labmate's templates are in pBluescript.

Could it be the vector's problems????

Please help me out! thanks!

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1 Answer

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More information is needed. Is the restriction site 600 bp from the transcription start site? What restriction enzyme did you use? The use of enzymes that create 3' overhangs can cause the polymerase to wrap around the template and start transcribing the antisense strand. Use an enzyme that creates blunt ends or 5' overhangs.

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