Question

Outline the steps required to close a eukaryotic gene using E. coli from mRNA derived from cells expressing the gene. Include how you would amplify the specific gene and how you would select for bacterial cells containing the cloned gene if the gene was inserted into a multiple cloning site within the lacZ gene on a plasmic. If you wanted to later express the gene using a eukaryotic expression vector and promoter how would you ensure the gene was in the correct orientation relative to the promoter?

Please answer all questions and in detail. Thank you very much.

Answer 1

Hi,

The following are the steps to isolate a gene using E. coli :

1. RNA Isolation: RNA isolation kits (Macherey-Nagal GmbH kit) can be used to isolate total RNA and is to be quantified.

2. cDNA synthesis: Reverse transcription PCR (RT-PCR) can be performed to synthesize cDNA using kit method (M-MLV reverse transcriptase kit).

3. The specific candidate gene can be amplified from first strand cDNA synthesized using gene specific primers (using PCR).

4. After PCR, the amplicons are to be resolved on agarose gel (gel electrophorosis).

5. The gel containing DNA bands are visualised under UV and documented using a UV-trans-illuminator.

6. DNA bands were excised and the PCR amplicons are extraced using kit mehod viz., Nucleospin Extract II kit (Machery Nagel, Germany).

7. These elued PCR product can be sored at 4⁰c for further gene expression.

8. The PCR fragment can be ligated into vector using kit viz., InsTAclone PCR cloning kit

9. E. coli cells are to be made competent by TransformAid bacterial transformation kit

10. After overnight incubation, Blue and White colonies will be formed indicating non-transformed and transformed ones

11. White colony (transformed) should be selected for plasmid isolation

12. Plasmids can be isolated from these transformed colonies using kits viz., GeneJET™ Plasmid MiniPrep Kit

13. Purified plasmids can be quantified using NanoDrop Lite and the can be sequenced using Sanger sequencing methods. The remaining plasmids (positive clones) can be stored at -80⁰c for further usage.

14. The homology of these sequences can be verified using NCBI BLAST.