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dcm-,dam- plasmid extraction

dcm-,dam- plasmid extraction - DNA Extraction Forum

dcm-,dam- plasmid extraction - DNA Isolation Forum. A forum specifically about questions on DNA extraction and purification.


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Old 06-29-2009, 08:52 AM
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Default dcm-,dam- plasmid extraction



Hey,

we have to transfer a kan resistance fragment to a zeo resistance fragment in a vector for cloning. This fragment can only be cut with BclI and EcoRV. Because of that, we have to make a dcm-, dam- vector.
After the transformation, we use machery-nagel kit to make plasmid DNA.
The problem we've got is that we have very less yield and when we do a digest the DNA is broken down.

We grow the vector on 37C for the plasmid extraction in 200 ml. We use also lysozym before the lysisbuffer. After neutralisation we centrifuge 15 min by 4000rpm. Than we follow the rest of the low-copy plasmid protocol.
We get concentrations of max 250 ng/l.

We do first the digest with BclI at 50C just 1 hour and than 1 hour 37C with EcoRV. After checking on gel, there's nothing but smear. Do we only put the DNA by 50C and 37C, the DNA stay stable.

What can we do to get a higher yield and DNA that is stable enough for digest?

Thx
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