Hi all
Another pKNG101 question. I have successfully optimized my restriction digests, but now I am getting multiple bands when digesting with SmaI. I have a suspicion that the plasmids stocks that were given to me already contain an insert, as the plasmid looks to be larger than 7kb (native plasmid is 6.8kb). The person who gave me the stocks previously digested the plasmid with SmaI as well to do his inserts. Ordering the plasmid again is unfortunately not an option. I was thinking of doing an inverse PCR to amplify the backbone of the plasmid around the SmaI in MCS in order to remove the insert, but for the life of me I can't find the sequence. I tried searching for the sequence of the MCS site itself, but the sequence of the plasmid it was derived from is also not available. Does anybody by any chance have it? Or have any other suggestions as to how I can obtain it? I was thinking of maybe designing primers that attach to the SacB and the StreptomycinAB section and sequence backwards with Sanger sequencing in order to obtain the sequence of the MCS between these two sections. Anybody have other suggestions?
After searching in google, I found these links may be useful to you.
http://www.sciencedirect.com/science/article/pii/0378111991905997
https://www.addgene.org/71298/
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