After double digestion of plasmid with Xba1 and HindIII for 4 hours, I run it on gel but unexpectedly each lane was full of smears and the bands were not clearly visible. What can be the possible reasons?
Perhaps the plasmid preparation still contains proteins or genomic DNA, which is digested, too. Does the plasmid show a single band when you use only HindIII or XbaI?
Perhaps the plasmid preparation still contains proteins or genomic DNA, which is digested, too. Does the plasmid show a single band when you use only HindIII or XbaI?
it will have smears naturaly as digestion is actually cutting out of DNA fragment and when u use 1 ug of starting conc then if you cut then the cut fragments would be in form of smears
How was the control (plasmid DNA in 1x restriction enzyme buffer but without restriction enzyme)?
The restrction enzyme buffer you used, are the two REs active in that buffer? Any star activity?
Hello,
try to lower incubation time and check the restriction buffer because too long incubation might caused the sample DNA to overdigested, and improper use of buffer can cause to star activity.
And i agree with Mr Michael, you should check undigested sample to see the quality of your sample.
the undigested sample is clear without any smears. the smears are only seen in case of digested samples.. The buffer is suitable for both the enzymes as I have checked it in the NEB side double digester.. in my opinion it can be due to enzyme binding with DNA, so I will treat it with 0.2-0.5% SDS, that might be helpful in isolating them from each other... as for as contamination of the plasmid with genomic DNA is concerned, I don't think so, as the undigested plasmid doesn't have any sign of that when seen on gel..
Thanks to all of you anyway
Is it same with the linear fragment, if you cut it with both of you REs? if you have done?
I'm having the same problem and wonder if I have genomic DNA contamination... I followed the miniprep protocol strictly, but I would accept any suggestion on how to address this issue
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