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High Molecular Weight DNA extraction and PFGE

high molecular weight DNA PFGE

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2 replies to this topic

#1 Nickiya

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Posted 08 May 2013 - 04:44 AM

Hello,

I'm currently using different techniques to extract high molecular weight DNA (up to around 120kb) from Streptomyces spp. and am finding the literature rather lacking in alternative methodologies.

Using a kit is unfotunately not an option.

Any tips and tricks would be greatly appreciated.

#2 Tiago Ribeiro

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Posted 09 May 2013 - 08:14 AM

Hi Nickiya,

I'm trying to make a BAC library at the moment and I followed this protocol - chapter 7 (http://wheat.pw.usda...f/BACmanual.pdf) to extract HMW DNA. The procedure worked well and I obtained a high quality and concentrated DNA. Have a look at that!

Do you have any experience in pulsed field electrophoresis? I've been experiencing a lot of problems in separating the PFG marker. Even modifying switch times/voltage, agarose percentage on gel and running buffer it still does not work. Either the marker does not run straight 1.jpg or separetes just a little tamanho clones 06-05.jpg , even below a high voltage and long run time. Any tips about that?

Regards,

#3 phage434

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Posted 13 May 2013 - 04:44 AM

What is your marker? Is the buffer cooled? The NEB yeast chromosome marker works very well in our hands. The lambda marker works less well.





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