From my experience, the key thing that makes the most difference is the concentration. Each entry clones should be at 10 fM, only the destination vector should be at 20 fM. Based on your formula, that looks to be correct for the entry clones. Total reaction volume should be 8 µL, not 5 µL. I would also try the following:

1. Check concentrations of each component again.

2. Check pH of TE buffer. It needs to be at pH 8.0.

After ProK deactivation as you said, I transform 3 µL into TopTen chemically competent cells. Let incubate on ice for 30 minutes. Heat shock at 42 C for 45 seconds. Let recover in 250 µL of LB or SOC medium at 37C for 1 hour (shaking horizontally). Plate onto Ampicilin plates. 

I don't normally get incorrect constructs. Most of mine are 80-90% correct. If you haven't used each of the entry clones successfully, I would also sequence each of them to make sure that the appropriate att sites are there.

Good luck!

Thank you Sataree. When you say the total volume should be 8ul, how does that work? Also, what formula do you use to calculate the destination vector concentration? My destination vector is 17kb+ Do you think this could be a hindrance? Please let me know. Many thanks.

Add TE as necessary to get 8 µL total volume. That is the volume that I use per 1 µL of LR clonase II plus. To figure out how many nanogram of each component you will need, use this formula: 

x ng = y fmoles x N (your bp) x 660 fg/fmoles x 10^-6 ng/fg; y=10 fmoles for each entry clone, 20 fmoles for your destination vector.

I have never used a destination vector that large. I don't know if that is causing a problem for you. I use pDestTol2CG2 and it's about 8 kb. See http://tol2kit.genetics.utah.edu/index.php/List_of_entry_and_destination_vectors for list of commonly used vectors. 

Hi Kunai,

In the past we had assembled the constructs using the 4 “entry clones” (in our case fragments) and the destination vector. Our destination vectors were 19.7kb or 9.6kb

Please attached doc file for details

Good luck

***** in our case it helped using the linear destination vector instead of the super coiled vector

Good luck

Hi Kunnai,

I agree with Zoia, we had to linearize the destination vectors before LR. The early vectors of the pGWB series require cutting with XhoI for example.

Good luck,

Maren

To linearize the destination vector you aim for single cutter in the ccdB/ Cam region

(between the "att" sites). 

As control cut the destination vector with other single cutter enzyme (to use as molecular weight marker for the linear form of your vector) and run on the gel.

Having this control will make it easier for you to  determine which is the linear form on the gel and  also if the digest is complete.Gel extract and try to avoid contamination with other forms of the plasmid. 

I also treat with SAP to prevent self ligation of the destination vector. 

Hello all, 

Presently I am also having a similar problem of not able to get any transformation when I tried to clone 2 entry clone into the destination vector. I have used LR clonase II enzyme. For multiple entry LR clonase II plus is used, but the protocol that I am using suggest LR clonase II is also good enough for assembly of 3 constructs. Do anybody of you have a suggestion, on how can I best use LR clonase II enzyme assembling 3 plasmid?.

Dear Maren and Zoia,

I am having issues with the LR reaction and I want to consult you two for advice.

My most recent attempt at the LR reaction was using a pGWB5 Destination vector (linear) + my Entry clone (supercoiled) and yet still no success.

Here's how I set up the LR:

Entry (53 ng/ul) x 2.3ul

Destination (71 ng/ul) x 1.7ul

TE (pH 8.0) 4ul

LR clonase plus mix: 1ul

---------------------------------------

Incubate O/N @ 25 Celcius

Do I need to linearize the Entry clone for successful recombination? Am I adding too much destination plasmid? If so would you suggest using 1ul of 70ng Destination?

Please any help/suggestions would be extremely appreciated.

Thank you!