Hello colleagues,
We are currently working on the MuLE system that was published on JCI (J Clin Invest. 2015;125(4):1603–1619. doi:10.1172/JCI79743.) . However, I'm having some technical difficulties on the L/R recombination, and I'm wondering if anyone could give me some suggestions on trouble shooting.
First, let me describe what we have done so far:
All vectors were from Addgene (Multiple Lentiviral Expression (MuLE) system - #1000000060). Vectors were purified by using Qiagen midi prep kit. Before recombination, vectors have been checked using DNA gel for the size. We also recently sequenced the vector to confirm that we used the right L/R arms in each recombination.
The recombination kit was Gateway LR Clonase® II Plus Enzyme mix (Life Technologies #12538-120). We diluted the vectors pMuLE ENTR CMV L1-R5, pMuLE ENTR CMV L5-L2, and pDEST-NEO to 150 ng/uL as described in the kit's protocol, and used 1ul of each vector and 5 ul of TE buffer, 2ul enzyme RT for 20 hours, then add 1ul of protease K, 10min 37 degree).
The transformation was performed by using Mach 1 competent cells, (4ul sample mixed in 50ul competent cells, 30mins on ice, 30s heat shock, 2 mins on ice and recovery for 1 hour before plated on LB plate with AMP 100ug/ml and Chloramphenicol 25ug/ml).
Competent cells and LB plates have been validated by using other constructs.
However, in my several attempts, I got either no colony at all or less than 3 colonies, which were all negative based on enzyme digestion results.
I'm wondering if there are any obvious errors in my description.
Thanks in advance for any suggestion and best,
Wei
First thing, use molar ratio of Destination vector:Entry clone1:Entry clone2 to 1:2:2. Use 150ng destination vector (pDEST-Neo) but then calculate entry clones required amount based on molar ratio calculation. Second thing, avoid using chloramphenicol to select your desired end product of destination clone as Ampicillin will only be the bacterial selection after LR reaction.
Hi Kumar,
Thanks for the reply. I appreciate it. I will calculate the entry vectors accordingly.
But why shouldn't I use chloramphenicol to select the recombination product? The destination vector (pDEST-Neo) carries resistant genes of both AMP and CHL. Do you mean that I should use only one type of antibiotic to reduce the stress to cells?
Thanks,
Wei
pDEST-Neo has chloramphenicol resistance gene along with ccdB gene between attR sites and Ampicillin in vector backbone. After LR recombination, cassette of ccdB and Chloramphenicol genes will be transferred to the byproduct of LR recombination..so you won't be having chloramphenicol gene in the final desired clones of pDEST. Hence, in your final product you will have Ampicillin selection only and you should use only Ampicillin antibiotic for selection of your desired clones.
You can read Gateway cloning in an excellent review by Katzen published in Expert Opinion in Drug Discovery (2007)2 (4), 571-589
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