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| DNA Extraction Forum DNA Isolation Forum. A forum specifically about questions on DNA extraction and purification. |
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| 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 37°C 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/µl. We do first the digest with BclI at 50°C just 1 hour and than 1 hour 37°C with EcoRV. After checking on gel, there's nothing but smear. Do we only put the DNA by 50°C and 37°C, 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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| dam , dcm , extraction , plasmid |
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