3 replies to this topic

[bio_kid]

Hi !

 

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

                                    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?

 

Thanks!

[Wunder]

Hi

 

In step one you should definitely search for the orthologous genes but then use this information to design primers for PCR. If you start with a restriction digest you will get a phenomenal amount of fragments most of which will not be your gene. PCR will amplify only your gene (if you design your primers right) and increase their abundance making the next cloning step a lot easier. As a tip design lots of different primers to try as they are cheap and some work better than others. Once its cloned you can use universal primers for your vector to verify your sequence - that way you get the entire PCR fragment in your sequence as the beginning and ends of sequences can be of low quality.

 

10kb is a really long fragment. As a back up you may what to consider using multiple primer pairs that amplify overlapping sections of the gene (if you can, it depends on what you want to do with it after DNA purification). If you don't know the internal sequence you can get universal primers for your species that may work with a gene specific primer as one of the primer pairs.   

 

Everything else looks okay but others may spot mistakes I haven't noticed.

 

Hope that's some help,

Edited by Wunder, 15 April 2015 - 03:19 AM.

[bio_kid]

What is your opinion on degenerate primers? I've read about these primers which are synthesized using softvers for sequence prediction based on homology between orthologous genes. Please correct me if I'm wrong. Do you think it is a good alternative? I just have to explain the cloning process, the later gene manipulation is unknown in my assignment. And I forgot to mention that the genome of an organism which gene I have to clone is not sequenced. I only know sequences of orthologous genes. 

 

Thank you

[Wunder]

Yeah degenerate primers can work I've even designed some myself (you can find lots of protocols and tips on how to do this online). Non-degenerate tend to work better so if you can find conserved sections of your gene where you can design primers I would do that.

 

If I was going to try and clone an unknown gene I would find all the orthologous sequences from related individuals (as closely related as possible, so preferably at the genus level) from NCBI or ENA. Then align them and search for conserved areas just outside my gene where I could design primers which would amplify my entire gene. Of course there will be some differences between the sequences so you could design degenerate and non-degenerate (based upon the most common nucleotides) primers. Also don't design only one primer pair I like to design at least three primer pairs from three different areas because then I can try them all at once and see which one gives me the best result rather than spend months trying to get a PCR to work with one primer pair.        

 

I mentioned what you were going to do with them after the cloning because if you were just looking at the sequence then amplifying and cloning overlapping sections of the gene is okay but it you want the entire gene (for transformation or something) then you will have to pcr and clone the entire thing which is really hard with long sequences but it can be done with patience and some optimising. Make sure your vector of choice can cope with 10k of sequence.

 

No worries

Edited by Wunder, 16 April 2015 - 06:56 AM.