advice on construct - possible problem with vector (Aug/31/2006 )
i've recently made a construct, and there may be a slight problem, and i need some advice.
the vector is, well almost boutique... it was made by another lab. I have a "map", which isreally difficult to read, and they haven't published the sequence... so i've pestered them about sending it to me, and they haven't been forthcoming with it.
based on their map, i put in a large promoter, and a large gene of interest. i've sequenced them, and they're fine.
i have sequencing primers that read in the vector (so from my promoter, i have a reverse primer that reads back into the mcs. from my goi, i have a forward primer that reads into the mcs and beyond). the sequence read that goes into the vector shows restriction enzyme sites completely different to what is on the map.
this construct is supposed to be used for transgenic mouse production. and i have to cut things out before little mices are born... the enzyme's they recommend to use... well, one cuts in 2 places, (on the "map" it only cuts in one... but i can see the RE site in my sequencing of the mcs.)
the other, doesn't cut at all (and it missing on my sequencing). but, when i cut the empty vector, it does what it's supposed to.
I don't want to have to start again.... i think my boss would kill me.
so, what would you do?
keep your chin up. similar things have happened to me, and unfortunately i had to make the vector myself. but it needn't be the case for you. sequence as mush of the vector you have as possible (if possible) and try to piece together exactly what it is.
the more info you ahve when you go to your boss, the better.
Also, not sure if you want to share info or not - but what is the vector - someone else (ie me) might have something similar that they can share?
edited because i pressed edit instead of reply.
It's a brand new family of vectors, that i have no idea how they were made. they have ECFP, a Sv40 poly A site and a Flp site for Flp-based predetermined site integration....
the mcs from my sequencing matches exactly with pBluescript SK+ and - (and a whole heap of others). the next 300 bp after that matches another page of vectors...
So long as the fragment i've cut has the ECFP and Sv40 poly A.... i'm laughing but if it doesn't....
i wish they would just send me the sequence.
well, i have really no financial interest, but i've sequenced a whole vector at MWG DNA facilities. I can send you details by pm if you want cause i don't want to make kind if advertising in this forum.
The results were done (and fine) in 1week.
I'm with Fred. Sequence it. You will thank yourself many times for having done it. You can do it yourself by choosing new sequencing primers at the end of good sequence you already have, and using them to continue the sequence. If you care about getting it exactly right, you should also make reverse primers and resequence in the reverse direction as well. You can move about 700 bp / round at each place you are anchored. Since you know ECFP or CMV sequence, you could start there as well, but you can get a minimum of 1500 bp / round, and it will only take 3-4 rounds, likely. Then, to spite your "colleagues" you should put it into genbank.
i cancelled the mice order this morning.
better nip the mistake in the bud now, then 12 months from now wonder why my mice don't have cancer. that's sounds so wrong.
edtited because i just broke tha bad news at work... i'm so dead. lovely to have known you all. tata
chin up - worse things have happened (although, at this juncture I'm sure you find that hard to believe).
I guess it is about minimising risk and loss - you did the right thing!
i'm so sorry for that bad news...
Hope you'll be able to continue soon;