I would appreciate some help on my assingment, I have an idea but I would like to hear your opinion since I'm still a uni student and I'm sure lots of you have more experience than I have
Size of an unknown gene is 10kb.
Some parts of an unknown gene have high homology with sequenced orthologous genes.
What is necessary for cloning process? Describe cloning method in details.
So my idea was:
1. Searching for a location of orthologous genes in plasmid genome library using FISH and isolation of these genes from library clone using restriction digest with two enzymes (restriction sites are surrounding each of orthologous genes)
2. Subcloning of digested genes into expressive vectors
3. Choosing the vector in which unknown gene product is expressed (I'm not sure about this because the product of the gene is not given in assignment)
4. Isolation of analyzed gene
5. PCR amplification: a ) designing forward and reverse primers complementary to conserved sequences of orthologous genes.
Each of these primers will have restriction site (for one of two used enzymes) and hybridisation sequence complementary to 5' (in forward primer) and 3'(in reverse primer) end of the gene
b ) PCR reaction
6. Verification of PCR reaction using electrophoresis on agarose gel
7. Isolation of analyzed gene using PCR Purification Kit
8. Choosing a plasmid vector which capacity matches size of the gene and which has two restrictiond sites for each of used enzymes (and antibiotic resistance gene)
9. Restriction digest of PCR product and recipient plasmid
10. Electophoresis of digest products on agarose gel and cutting off the bands which positions on the gel match the size of plasmid and analyzed gene represented on the ladder
11. DNA ligation of analyzed gene and recipient plasmid
12. Transformation of a host cell (E. coli)
13. Plantation (I'm not sure I'm using the right term here xD) of host cells onto medium containing the antibiotic
14. Overnight incubation
15. Identification of bacterial colonies
16. DNA purification (separation of plasmid DNA from bacterial culture)
17. Repeat steps 9 and 10
What steps should I change? What sould I add and what should I remove?