Genetics – HSCI 10022|
Assignment # 1|
Assignment Due Date: Tuesday, March 8th 2011
Prepared By:Jelena Komazec Student Number:000012828 Prepared For: Dr. U. Vivegananthan
i) The enzyme that would be used to cut the vector would be SalI along with adding the CIP. The main reason that you would cut with the particular enzyme Sal I is because the beginning of the vector sequence there isn’t a stop codon, so but cutting it with Sal I than both the vector and the protein will be compatible. There however is going to be a function of the CIP. CIP is a very important aspect of the whole process because once the vector is opened; the CIP is going to be able to remove the phosphate groups which are located on each end. Therefore by removing them there is no way for the vector to be able to rebind to itself. ii) The two restriction enzymes that you would have to use in the case of the protein is the Sal I along with Xho I. The reason that you would be able to use these two enzymes is because even though they aren’t the same enzyme they are still going to be compatible with one another. The beginning of the vector sequence is going to be cut with the Sal I and the protein is going to cut with Xho I. Than both sides are going to rejoin and make that appropriate sequence. There however isn’t going to be a CIP used because if the CIP were to be added than it would be unable to ligate or bond. iii) The enzyme that would be added to ligate the cut vector and protein X insert together would be T4 DNA ligase PART B.
5’ .... CAT CAC GTG AAG CCT GGA TTC G/TC GAG ACA AGC .... CTG CAG TAA ....3’ 3’.... GTA GTG CAC TTC GGA CCT AGG C/AG CTC TGT TCG .... GAC GTC ATT .... 5’ PART C
The cloning in the particular sequence would be considered not directional. I was able to establish this because if you were to flip the protein the other way, which would mean going from the Xho I...