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metylated cloning vector - restriction digest of the insert (Mar/29/2011 )

Hi all,
I am trying to clone a 3.5kb PCR produt into 5.3 kb bicistronic iRES contg cloning vector which is methylated (clontech)The vector is pIRES2-AcGFP1. The vector and the insert after the cloning reaction were transformed into DH5alpha cells.The cells are grown on kanamycin (50ug/uL)LB plates. Got far too many colonies . of these 20 colonies were picked up randomly and grown further in LB contg Kanamycin for 16 hrs. The plasmid was extracted using Qiagen columns. The purified plasmid was run on 0.8% agarose gel. To my surprise I get a distinct band at 3.5kb and a very faint band at 9kb. I am not able to understand this. This is the case with all the 20 colonies i picked up. They all are showing the same pattern. A faint 9kb band and a prominent 3.5 kb (which happens to be the size of my insert)band.

Q. wht this 3.5 kb band could be?
Q.Since the plasmid is methylated does tht mean no0w the band of my interest is also got methylated and that do i need to use a methylation sensitive R.E?
Q. is it possible tht a plasmid can release its insert? which is very unlikely, but still a doubt


Have you cut your plasmid prep with some restriction enzyme? If not, then the detected size of the supercoiled plasmid can be very different from the predicted one. Cut the plasmid with a RE (for example Nco1) and check the bands of your digested plasmid.
If your plasmid prep comes from a methylation-capable strain of E coli (which is most likely the case), then your plasmid and insert are methylated. Very few RE are methylation-sensitive (Xba1 for example is inhibited by methylation), so you don't have to worry about this.
Which RE sites have you used for cloning? If you used the same for 5' and 3' ends, then you may get self-ligation, and lots of empty vectors. I would suggest you to use different RE for cloning, and a gel purification to clean up your plasmid before ligation.


Thank you very much for the reply. No I had not used any RE digestion during plasmid purification.
That means I will have to gel purify the plasmid and then subject it to RE digestion.Will try tht.