An expression vector has a start codon ATG, and straight after it a BamHI site, GGATCC, which has 6…
Contributed by StemPy LabTry it on paper first. A hint, the key idea and the worked solution are here when you need them.
An expression vector has a start codon ATG, and straight after it a BamHI site, GGATCC, which has bases. The coding sequence of the gene is to be joined after the site, so that its first codon is read in the same frame as the ATG. A student designs a forward primer with a clamp, a BamHI site, two extra bases AT by mistake, and then the first codon of the gene.
- How many bases lie between the ATG of the vector and the first codon of the gene?
- What is the remainder when you divide this number by ?
- Is the frame kept? How could she repair the primer?
Need a reminder? The key ideaShow
A primer has 18 to 25 bases, 40 to 60 per cent G and C, and a G or C at the 3 prime end. , and the annealing temperature is about 5 degrees Celsius below it. Extension takes about 1 minute for each kilobase. The number of bases between the start codon and the insert must be a multiple of 3.
The worked solution
Have you tried it?
Work it out on paper first. It is fine to get stuck or make a mistake: that is how we learn. When you are ready, go through the solution one step at a time, and check each step against your own.
Given. The site has bases. Two extra bases.
Plan. Count the bases between the start codon and the first codon of the gene. The number must be a multiple of .
Step 1. (a) bases.
Step 2. (b) is with a remainder of .
Step 3. (c) The remainder is not , so the frame is shifted. Every codon after the site is read wrongly, and the protein is not the one that is wanted. She can repair it by removing the two extra bases, so that only the bases of the site lie between. Adding one base would also make it .
Answer: bases; a remainder of ; no, the frame is shifted, and she should remove the two extra bases.
Watch out. The clamp lies outside the site, in front of it. It is cut away by the enzyme, so it does not count.
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