I am having issues with the LR reaction (3 entry vectors + pDest). I get no colonies apart from when a reaction of 1:1 ratio is used, which gives me around 1-2 colonies, but it turned out to be incorrect sizes.

I have done the following ratios of LR reaction so far:

10 fmol of each entry and 20 fmol of destination

20 fmol of each entry and 20 fmol of destination (gave 2 colonies, but both incorrect)

30 fmol of each entry and 10 fmol of destination

LR Reaction Conditions

All the reactions are done in 5 ul, but I have tried in 10ul too for the 1:2 reaction.

The reactions contain, the entry vector, destination vector, TE buffer (pH 8) and LR clonase II.

25 degrees for 16 hours then 0.5 ul proteinase K for 10 min.

Transformation

Transformation into OneShot Top10, also tried in DH5a cells

30 min Ice, 42 degrees 30 second heat shock, recovery in 250 ul SOC for 1.5hrs, then plate 250ul on Amp resistant plates.

I have also tried with p5e and destination vector linearised, still no colonies or 1-2 incorrect colonies.

Original Plasmids

All the entry and destination vectors have been restriction digested and checked by gel for correct size, then all the att site regions of the vectors have been sequenced. Performed electronic LR reaction using the ApE software which was able to perform the LR reaction and generate the correct final vector, therefore, I don’t believe there is any issues at the sequence/design level.

The stock concentration of the vectors are around 400ng/ul, I dilute it 10x (in water) to around 40ng/ul and use that for all the reactions above.

Vector sizes are:

P5E - 5068BP

PME - 7648BP

P3E - 4088BP

pDest-tol2 - 5883BP

Simplified formulae that I used for the calculations are: amount in fmol x length of plasmid in bp x 0.00066

So as an example, for 10fmol of the P5E entry vector (5068bp), the amount used was 33ng.

Ideas to test?

1. The plasmids were extracted via GeneJET Plasmid Miniprep Kit, I read in places that purelink should be used, but not sure how much of an impact that would really make?

2. I'm next planning to make a pre-mix of the 3 entry in TE buffer, then take 1 ul of that (10 fmol) + 20 fmol of pDest + TE up to 4ul and 1 ul of Clonase. Again not sure if that would make an impact.

3. Also, is it worth increasing the amount of DNA used to around 60ng or more per entry and adjust the other plasmids to have correct fmole ratio?

Thank you in advance for any suggestions 🙏