Using the gene fragment and vector shown below, you digest both with Hind III, a
ID: 259837 • Letter: U
Question
Using the gene fragment and vector shown below, you digest both with Hind III, and then add ligase in order to clone the gene into the vector. Since the insert can ligate into the vector in either direction, describe how you would determine the orientation after the cloning step. Show your data and state how they support your conclusion. HINT: restriction enzyme digestion and gel electrophoresis.
Hind III EcoRI Hind III EcoRI 5 overhangs EcoRI Eco RI Insert ? 900 bp Hind III Eco RI Vector 3000bp + alkaline phosphatase EcoRI mp Hind III 300 bp 600 bp Insert mp Hind IIl DNA Ligase 400 bp 3000bp gl II Recombinant DNA mpExplanation / Answer
Answer to determine the correct orientation of tha insert can be determined by many method First method is blue white screening method in this vector consist of LacZa gene in the multiple cloning site. Also using the E.coli strain which is LacZdelta15 mutation. If cloning occurs then white colony will be in plate while there will be blue colony.
2nd method is colony PCR, so colony of a bacteria taken and PCR is done using the primer with having insert sequence and vector sequence if you get the exact band of DNA then your insert is oriented in right manner.
3rd you can also done by digesting the vector and after it running on the gel electrophoresis then the band will be of your expected size with insert
Related Questions
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.