I've been working on making a working gateway plasmid with LR clonase. I've been following the protocol step by step, but I don't get colonies.
I use the LR clonase II plus from Thermofisher. The plasmids that I use are from the addgene kit (pMVP and pMAGIC Cloning Systems). I dilute the plasmids with TE buffer to a concentration of 10 ng for the 5', 3' and middle entry clones and 50 ng for the destination plasmid. The transformation is done with Top10.
If you have some ideas where the problem could be, I'd really appreciate it.
You can try calculate the right amount of pEntry and pDest you need for the LR reaction by using this formula:
xng=yfmol*N(bp)*660fg/fmoles*10-6ng/fmole
you should use 10fmol for pEntry and 20fmol for pDest
Ligate for at least 3 hours at room temperature.
Another thing you can try is to use 5 ul of ligation to transform One Shot™ TOP10 Chemically Competent Cells and plate all 250ul.
Yeah you can stimate the mass you need also by ligation calculator (NEB). Then there are several tricks.
1. You can linearize the donor (your first plasmid pEntry) just in the resistance site.
2. Increase DNA 50 ng and the amount of pEntry to get 1 pDest: 3 pEntry
2. I have my rule of "the 2":
2 h 22 min at 22C for recombination then 2.2 ul recombination product to 22.2 ul cells (one shot top10 should be ok ). 43 C for 45 sec and recovery of 2 h 22 min at 37C. Finally plate 222 ul on a plate containing the desired antibiotic (remember antibiotic in the pDest plasmid ). Please check if your pDest has the lethal gene in that case you will need a specific e coli strain.
Regards
Noe Baruch
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