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Help: Best way to disrupt blood cells to generate blood debris and gDNA to be us

Blood debris Detergent free gDNA

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4 replies to this topic

#1 Fluoresce

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Posted 09 March 2013 - 09:40 PM

I am trying to design an experiment to test a microfluidic device with a control sample with an exagerated amount of blood cell debris (i.e. more than 50% of cells destroyed, all cells included) and gDNA. I need to keep this sample detergent free and outside particles free (i.e. can not have beads from bead beaters shed particles in the sample) for the purpose of being tested with a microfluidic device.
I have thought of freeze-thawing, sonication and dounce hemogenizer...However, I have no access to a french press. Does anyone have any suggestions or recommendations for any of these mehchanical methods? Will I be able to disrupt the nuclear membrane with any of these methods without applying detergents? I would really appreciate any help or suggestions.

Thanks!

#2 bob1

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Posted 10 March 2013 - 11:43 AM

Freeze/thaw should work well - the ice crystal formation is enough to lyse the cells fairly effectively. I would do about 3 cycles for complete lysis.

#3 Fluoresce

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Posted 10 March 2013 - 11:58 AM

Hello Bob...There you go...I always get the best answers from you. Thanks a lot. How long would you keep the cycles going though?

#4 bob1

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Posted 10 March 2013 - 12:12 PM

Basically you don't need to do any more than ensure that the blood is fully frozen and then fully thawed. The quickest way to do this is use a dry ice/isopropanol bath (dry ice pellets in isopropanol, take care with tube labelling, this will remove most pen written labels) and a 37 degree bath, dunk the tubes into the baths alternately. You could also use liquid nitrogen, but it is a bit more dangerous, isopropanol at -80 is also effective, but warms up rapidly unless you work in the freezer.

#5 Fluoresce

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Posted 10 March 2013 - 06:40 PM

Awesome! Thanks so much for your help. I really appreciate it. I'm going to try the dry ice/isoproponaol/37 C bath...This defintely is going to happen first thing when I get my hands on some blood this week ;) Thanks again and have a great week ahead!





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