need some advice on introducing a marker - (Feb/05/2006 )
Perhaps someone here can help me with a few questions concerning a homework.
The task is the elaboration of a procedure way for the insertion of a selectable marker suitable for linkage mapping in S.cerevisiae. It is to be inserted between ADE5,7 and LYS5 at the chromosom VII.
Now my idea would have been to manage this by use of a disruption-cassette which contains short homologous flanking sequences of the gene to disrupt and in between the marker gene, I thought of NeoR or KanR. For the gene to disrupt every gene is suitable that produces a viable phenotype, right?
Questions emerged by the fact that the Professor explained a problem similar to this but he used a Yeast integration Plasmid instead of a disruption cassette. The YIP contained the marker and the gene, which is in the desired range. Then the Plasmid is to integrate by homologous recombination at the desired place including the marker.
now I don't know whether both possibilities would work or whether there are reasons against one possibilty. Isn't it faster to construct the the disruption-cassette instead of the YIP?
So how are you creating this disruption cassette without a plasmid?
Your english is very difficult to understand. If I understand your english, the principle for both approaches is the same.
Strictly concerning your last question:
Difficulty of plasmid or disruption cassette construction can vary depending on method of construction or cloning. I wouldn't say one is easier than another. However, I've done a couple thousand gene disruptions in yeast, fungi and bacteria and one of my favorite (and quickest ways) to build a cassette was using a 3 part pcr method (crossover PCR, etc.)
Now if I understand what you're describing - there can be several different outcomes of integration when using an integrating plasmid or disruption cassette (with flanking homologous regions). The plasmid with a single homologous region will do exactly what its called integrate, if that's what you need. The cassette your describing will delete the chromosomal region unless your flanking regions are picked from sequential chromosomal sequence.
...does that make sense?