Transformation and miRNA
Simoe Goss
Wednesday, 04 February 2009 23:12 UTC
Hello,
I am a Master student and my project involves miRNA research.
My biggest hurdle so far involves transformation.
I seem to be struggling to get any colonies to grow on my agar plates.
I am using a pGL-3 control plasmid, where I inserted my miRNA target sequence using a single restriction cutting site using XbaI.
I added ligation reaction to the DH5-alpha cells. I than put the reaction on ice for 30 minutes, than heat shocked it for 45 seconds at 42 degrees Celcius.
After that, I added LB and left the bacteria to grow overnight at 37 degrees Celcius. I than spread the colonies onto the agar plates.
I use 100 microlitres of Ampicillin/1ml of 1.5x Agar with LB
I use 3 controls, one control without the oligo/primer, positive control (using uncut pGL-3) and negative control (cut pGL-3).
The results I have been getting is as follows:
- postive control shows a few colonies, other controls no growth and no growth for the miRNA target gene
or
- no colonies on either plates including all three controls
Can someone please give me advice on this? It would be extremely appreciated
5/2/09:
I would also like to state that I have tried various competant cells including DH5-alpha (fresh and frozen) and JM109 cells, and ordered fresh Ligase, for the ligation reaction.
Thanks.
Updated 04 February 2009 23:17 UTC
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Replies
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How big is your target sequence? Why dodn’t you synthesize the MRE as two single strnded oligos (complementary) in a ligation ready form, ie., by adding open restriction sites on either sides, and then anneal these single stranded oligos first and then cut your vector, purify by PCR clean up kit and ligate with the previously made (annealed oligos) MRE. This should work. Atleast this is the way I make this.
Regards
Bernard -
Hey, I’m also in the same position, pursuing a masters on miRNA.
For transformations, our lab’s been using Gateway Cloning
It’s really effective, and just requires a PCR product of your gene with special sequences at the ends, entry vectors and expression vectors, no restriction enzymes. This was passed down to me by the postdocs and phd’s, who had trouble with restriction enzyme cloning.To break it down roughly, if you’re interested, what happens is that you would design gene specific primers for your gene with homologous sequences to the entry vector at the ends of those primers (dubbed “attb” sites). Do a PCR reaction and gel purification. Then mix the PCR product with an entry vector and BP clonase in a BP reaction, and by homologous recombination your gene with flanking sequences homologous to a specific gene in the vector will be inserted into the entry vector. To get the gene into an expression clone, which you can use directly to transform an organism with, mix the entry vector, with the inserted gene, together with a destination vector and with LR clonase enzyme. Both vectors will have special “att” sites that make it possible for the gene to be inserted into the destination vector by homologous recombination. Of course, you’d also have to plate and select for E.coli cells with the various vectors. DH5-alpha cells work really well with Gateway Cloning transformations, I usually get 100s of colonies after 16 hours of incubation, and at least 50% of the minipreps yield vectors with my gene in them. I do an restriction enzyme digestion specific for the gene and vector to confirm.
Good Luck!
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