hello, i would appreciate any advice on my issue
i am doing two similar constructs in the same time - i am trying to put two mammalian promoters in an expression vector containing gfp.
i previously cloned the promoters in pcr-topo and sequenced the plasmid, they are ok.
the vector i am using contains cmv promoter which has to be replaced by mine. So, i cut off the promoters from the topo, i cut off the cmv with the same restrictase SpeI (no possibilities for double digestion, unfortunately). I dephosphorylate the vector since the ends are similar and i don't want it to religate; i run everything on gel and bands seem perfect. i purify them with the qiagen kit and set up ligations at 1:1 1:3 1:6 and 1:10 ratios with t4 ligase by neb overnight at RT (buffer aliquoted, no multiple freezes and thaws). I know that RT works fine because I performed similar ligation under these conditions (again speI and inserts and vector of the same size) and it gave me about 100 nice colonies.
This time, however, i got only 4-5 colonies for each construct only in the 1:6 plate. Positive control - uncut vector was perfect - hundreds of colonies, negative controls (cells only, cut vector +ligase) gave nothing. anyway, i did a miniprep on all of them and digested the plasmid with xbai and ecoRV /to check for orientation, ecoRV cuts in the end of both my inserts, and xbai is in the vector/. Again i used those enzymes together before and i know they work just fine.
Surprisingly, I got absolutely mysterious bands that didn't match any expectations - any sizes, multiple inserts, self-ligated vector, etc. The control of just vector with the enzymes gave me one band (i.e. only cut at the xbaI site, ecorv is in the insert) of the correct size. I expected either 1000&7000 or 3000&5000kb bands, but i got different ones between 1500 and 3500 roughly, and three instead of two for most colonies. however for some i got one band of >8kb. The pattern wasn't the same in all colonies for each construct, but some matched, for example for one construct two of the 5 plasmids gave me one pattern, 2 others - another pattern, and the 5th - something else.
I thought it was just a weird contamination, repeated the whole experiment from scratch, but again got weird very few colonies and the plasmids gave 2 or 3 bands after the digestion, with different than the previous but again wrong pattern. I tried different restrictases - again one in the insert and one in the vector - this time i got only one band at about 3000
Obviously something with the ligation is wrong, i should get more colonies, but despite this what can be the reason to observe those strange bands from the plasmids i get ?
Any ideas? Thanks
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