problem concerning PCR Test after disruption and yeastgenome DB - (Mar/07/2006 )
The task was to (theoretically) insert a Marker on ChrVII at a Position between ADE5,7 and LYS5. I managed it by using a disruption cassette for CUP2. The problem is that for the PCR-test to check if it has inserted at the correct position (i know it would by possible by adding Cu2+) I need to have an Oligo for CUP2 out like on the picture:
CUP2 is located between 191135 - 191812 so the primer (it should be about 18 NTs long) could have the sequence from 191117-191135, right?
Now at the Yeast Genome DB i just can find the sequence of open reading frames but not the sequence in between them.
Could anyone give me a hint about this or tell me how to get to the correct CUP2 out sequence??
which yeast? blast has a few types...is it possible for you to use blast for this?
s.cerevisiae - ok i will try it with blast - i just dunno what and in which field i should type in to get my results...well i'm trying to play around a little bit....
this is how I would do it...for your needs, you don't need to add a bunch of filters
type your oligo probe sequence into 'search' box at the top
under 'format' and 'advanced' settings, select your organism...ignore the rest
click 'blast' button
when new screen pops up, click 'format' button
your results will show up soon, usually within several seconds
does this help?
umm it worked by searching part of the cup2 sequence here: http://www.ncbi.nlm.nih.gov/blast/html/search.html
then clicking on retrive sequence and after that changing the url manually to 191117 - 191134:
not very elegant...
but for the way you described i would need to know the sequence of my probe right? But how can i know my oligoprobe-sequence when i don't know the sequence on the Chromosome?
I mean i just have the Position and Sequence of CUP2 but not the Sequence out of the CUP2 ORF... - on the picture i put online in the first post (i got it from my professor) it seems that CUP2-out-Primer is kind of before the ORF...so the only thing i know is that the primer needs to be the homologue sequence to the positions before 191135....and i must admit i don't really know much about primer design, as we haven't been tought of the practical aspects of PCR...
oooohhhhhhh I see what you are saying.
sorry, I didn't realize you didn't have your primer sequence. that doesn't make it easy for you does it?
actually, then, I would still do the blast, but use the CUP2 sequence from your yeastDB immediately downstream (at the beginning of the ORF) as your search parameter. look for contiguous sequence on the chromosome to find your upstream region after the blast search. you should be able to pull up the sequence for the gene and a bunch of stuff on either side that should tell you what you want to know
oh, by the way, your link sent me to a bunch of wierd stuff that you probably didnt' intend...I think our computers are not playing together well
have I helped you?
oh cool now it worked - thank you for the help!!
Yeah the problem about the primer sequence is that the only information I have about it until now is, that it has to be longer than 25 nucleotides and then the PCR should work. I'm just wondering about all the primer design functions you can find everywhere - it seems not to be that easy...well at least I've almost done most of my homework now...thank you again
by the way that's the picture - there's still a litte mistake - the one ALR1 because i just "overpainted" it for my purposes...