Hi,
I have been trying to clone an about 6kb large gene in to a vector that later is gonna be used for mRNA expression zebrafish.
We have been using the pCS2+ vector as it was provided by colleges with experience of mRNA expression in zebrafish. But it is not going very well. I have been trying three different kits for the ligation, with many different ratio of 1:2 to 1:10. But still no insert in my vector. I would like to try with another vector that can take my huge gene but is still suitable for expression mRNA expression in zebrafish - any suggestions?
Also, if you are sitting on an alternative design on how to clone this huge gene into my vector Im all ears. Im thinking about doing it by cloning several smaller pieces of the gene, one by one, what do you think about that?
Thank you!
Sanna
Hi Sanna,
I would suggest trying Gibson assembly instead of ligase. It is significantly more efficient. For designing the primers with overlaps (for the insert) use a program like Benchling or Genome compiler. If you recover empty vector you should additionally use two or even three restriction enzymes and phosphatase for linearisation. If the vector was cut only by one enzyme without phosphatase it will be re-ligated without insert in the ligation. I also find that purification of the fragments by a small column prior to ligation can be advantages.
regards
David
Hi Sanna,
pCS2 can definitely take 6kb without problem. I have used it in the past for a 7kb transcript. The problems seems to be with cloning. Are you PCR amplifying your insert or sub cloning it from another vector. If you are doing PCR, you may want to try Tol2 kit to assemble the construct.
http://tol2kit.genetics.utah.edu/index.php/List_of_entry_and_destination_vectors
Tol2 kit is a modular assembly kit that most fish labs have. You can clone your promoter, reporter and polyA in three ENTRY clone which can then be recombined with DESTination in correct order to give a expression plasmid. The promoter/polyA plasmids are already in the kit so you just need to clone your gene.
Good luck,
Sandeep
HI Sanna,
Try this pcGlobin2, optimized for zebrafish
http://www.ncbi.nlm.nih.gov/pubmed/15179057
Good luck
Sateesh
Hi David, Sandeep and Sateesh,
This was all very useful information for me. I will look it up more closely and get back to you on what works. Thanks alot!
Best regards,
Sanna Gudmundsson
FYI,
The project was put on hold so I therefore do not know what worked better. However I got another usefull tip along the way of cutting my fragment in several pices and then cloning a smaller parts one by one. Similar as suggested by David Ritchter.
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