checking the direction of the insert - (Oct/05/2006 )

Hi

How do I check that my insert is in the correct orientation in my promoter construct.

thanks

SY

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I look for restriciton digests that will yield different fragment lenghts, depending on correct or incorrect orientation.

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1. Try expressing it in 10 ml of culture. Grow up for an hr or so from an overnight culture, add IPTG, express for an hr, spin down, lyse cells and run an SDS-PAGE gel. If it's there, you'll see it at the right size.

2. Do a PCR using a primer from the vector (like a sequencing primer) and the revevse primer for the insert. If it's in the right orientation, you'll see a product (assuming your reaction is behaving itself). Clearly you have to make sur ethe primers will work together; if they're not exact, doda touchdown PCR to fix any slight problems with Tms.

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Hi,

You could also do sequencing using the seq primer specific to the promoter you're using, e.g. T7 etc.

Bests

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easy way : restriction digest

time consuming way: sequence it.

I would sequence it if any doubt appears.

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enzyme for the screening and sequencing for confirmation

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