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please urgently help: after phenol/chloroform extraction, my plasmid vector beca


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#1 veer2

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Posted 28 December 2009 - 11:34 PM

please urgently help: after phenol/chloroform extraction, my plasmid vector became smear on agrose gel

I am working on molecular clonning and fall into trouble. I plan to double digest my plasmid vector with SnaBI and KpnI. Due to the buffer system is incompatible for these two enzyme, I decide to do sequential digestion. Firstly, I digest 3ug plasmid with SnaBI for 1.5hr (50ul reaction volume), after the digestion, I took 5ul solution and ran on 1%agrose gel to confirm the digestion. Very clear one band could be detected and it is linear plasmid. Secondly, The rest 45 ul solution was used to phenol/chloroform extraction and dissolved in 10ul H2O finally. Thirdly, I cut the above 10ul DNA solution with KpnI (30ul reaction volume) for 1.5 hr and ran agrose gel to recover the DNA. But, it shows SMEAR on gel.

To confirm whether KpnI brings the problem. I repeat the experiments, but cut 3ug DNA with KpnI firstly for 1.5hr (50ul reaction volume), after the digestion, 5ul for running gel. And also very clear one band could be detected. The rest 45 ul solution was used to phenol/chloroform extraction and dissolved in 10ul H2O. Then, I cut the above DNA with SnaBI (30ul reaction volume) for 1.5hr again and ran agrose gel. But it show SMEAR again.

From above, it seems that the DNA smear appears due to the phenol/chlorofrm extraction step?? But, I do not know what is the reason? Ethanol rest after the extraction? but will it lead to DNA smear? or the solution for phenol/chlorofrm was contaminated by Dnase? but, my colleagues used the same solutions. And it is okay. Any advice?? What is the potential reason will lead to DNA smear in the procedure of phenol/chloroform extraction??

Thanks a lot in advance

#2 phage434

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Posted 29 December 2009 - 06:40 AM

I would definitely be doing a double digest with these two enzymes. Check here: http://www.neb.com/n...tCalculator.asp for recommendations. In this case, buffer 4 with extra KpnI. I suspect your problem is inadequate purification after the extraction. Do you do a chloroform only extract following the phenol/chloroform extract? Do you wash the pellet with 70% ethanol after spinning the pellet down? Do you let the ethanol evaporate prior to resuspension? Any of these could cause a problem. Also, you are digesting in too high a concentration. 3 ug in 30 ul is five times more concentrated than I would use (1 ug per 50 ul). This can cause the purity problems to be more severe, since a larger fraction of the reaction volume is "purified" product from the previous stage.

#3 jah

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Posted 29 December 2009 - 10:02 AM

I don't have much to add to Phage's suggestion, but could the smear be due to overloading the gel? A picture of your gel would help rule this out




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