Getting smear problems in plasmid run - (Sep/29/2007 )
Hi everybody,
I am cutting pcDNA Hygro by Not I enzyme (NEB) using Buffer D(Promega) but everytime I get the smear instead of a single band as Not I has 1 cutting site in pcDNA.I also ran a sample without Not I but only Buffer D but to my surprise I got 5 bands.I am very much perplexed.I have changed the autoclaved water,Buffer,enzyme and incubation time but still getting the same smear patterns.I havent seen the problem with pcDNA Hygro only as there was no smearing but only when I treated with other enzymes and reaction mix.If anybody can suggest me to solve this problem.Thanks in advance.
Enjoy Science. 
This indicates a probable nuclease contamination from the bacterial host or the
reagents used.Work under sterile conditions, wear gloves, and do not re-use tips.
could you put a picture up? Pictures are a big help.
ok generally smearing in DNA after restriction may come from :
*genomic DNA contamination (smear in high mass)
*nucleases(global smear) but you may see bands
*RNA present in the preparation (smear at bottom of the gel)
*star activity (global smear)
And i agree with pernesblue, may you please post a picture?
*genomic DNA contamination (smear in high mass)
*nucleases(global smear) but you may see bands
*RNA present in the preparation (smear at bottom of the gel)
*star activity (global smear)
And i agree with pernesblue, may you please post a picture?
Thanks a lot for your suggestions.I am posting a picture,hope it works.I did not get smear while running the vector only.The problem occurs when I cut it with the Not I enzyme.No,the Not I enzyme does not seem like contaminated because when I used it to cut pBLCAT3,there is no smear.
Did you put alot of your plasmid for digestion? how much did you put in for digestion in ug? You had your band there, but smearing. Can be caused by high conc of plasmid used. incomplete digestion in other word.
I am using 1ul of the plasmid DNA for cutting with Not I.I have not measured the amount of pDNA.I extracted it using commercial kit and the total volume was 50 ul.I used o.5 ul of Not I enzyme and incubated for 2 hrs and next time 6 hrs.You can see the gel run result in the attached image.So what do you think now?
Hi
Check for conc of Not1 that has been used and try using 1U for 1ug of DNA.Some times the DNase contamination might causing the degardation.Do another round of Phenol:Chloroform extraction of DNA and then digest for 2hr at 37 degrees.This may solve your problem
how much is your DNA plasmid in term of ug?