Question

Draw a diagram or create a concept map that explains the relationships in the appropriate order of the techniques shown below to create a sequential experiment for the synthesis of a recombinant vaccine for the Ebola virus.

I ordered it as follows:

1. E. coli host

2. Human insulin gene

3. Restriction enzyme

4. Probe

5. Gel electrophoresis

6. Southern blot

7. Primers

8. Genomic library

9. PCR

10. Plasmid expression vector

Please let me know if this is correct. If not, please list the correct order and explain. Thank you!

Answer 1

Above steps of the Synthesis of a recombinant vaccine for the Ebola virus is correct.We can see guidline for the molecular cloning in below picture.

Troubleshooting Guide For Molecular Cloning Vector choice Improper replication Absence of: An Origin of replication RE recognition site Selectable genetic marker Gene for screening • Methylation Salt and PCR component inhibition • Wrong buffer • Short incubation . Too little enzymes DNA fragment preparation Incomplete RE digestion • Digested DNA as smear • Extra bands on gel • No PCR fragment amplification Wrong: • PCR primers Annealing and extension temperature • Mg2+ concentration Buffer pH • Cyclic condition Recombinant DNA creation (using DNA ligase) Maximum ligation mixture Insufficient ligation, phosphorylation and A-Tailing Ligated DNA appearance as smear • Non-viable cells GOI toxic Use of wrong heat-shock protocol • Presence of PEG • Incorrect antibiotic and its concentration Insertion of Recombinant DNA into host No transformation: • Improper temperature • Storage and handling . Poor antibiotic resistance expression • DNA impurities and excess Constructed DNA: Too large Susceptible to recombination Colonies w/o plasmid: Insufficient antibiotic Satellite colonies selection Clone screening Colonies with wrong plasmid: • Recombination of plasmid • Use of incorrect PCR amplicon • Presence of internal recognition site and mutation sequence