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Loss of DNA after restriction digestion cleanup


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#1 chn09

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Posted 20 January 2014 - 10:42 AM

I had set a restriction digestion of pUC19 with Kpn1 and the plasmid was linearized. I wanted to cut the plasmid with EcoR1 and therefore i precipitated the DNA with 3M Sodium acetate pH4.5 and ethanol. I checked the plasmid DNA concentration on gel, loss of DNA was very evident. I set the EcoR1 digestion with the precipitated DNA and now only a faint band can be seen.

 

1. Is 3M Sodium acetate ethanol a good method for plasmid DNA precipitation? Is there any good method that gives a good DNA yeild (Ive heard of ammonium acetate but dont have the protocol for it)

 

2. Which is a good method for extraction of DNA from agarose gels (Phenol chloroform treatment ?

 

i dont have spin columns/ gel extraction columns for plasmid/PCR purification

 

thanks..



#2 jerryshelly1

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Posted 21 January 2014 - 08:18 AM

1) 10% sodium acetate in EtOH is a good precipitant. If you are starting with a small concentration of DNA, you can add glycogen to assist in the precipitation. Allow your DNA to precipitate at -80C for 30-60min for good results.

 

2) If you do not have any columns, you can do phenol cleanup for agarose. Google a protocol (they are all very similar and easy). You basically melt your agarose in a buffer (Tris) and the proceed via a regular phenol chloroform cleanup.



#3 phage434

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Posted 21 January 2014 - 08:59 AM

You should be able to cut with KpnI and EcoRI at the same time. Fewer steps, fewer times to lose your DNA. You can't heat kill the KpnI, so you will need to do some sort of cleanup.






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