MultiSite Gateway® Three-Fragment Vector Construction Kit - FAQs

Can I perform the single-step protocol for the BP/LR Clonase reaction using BP Clonase enzyme and LR Clonase enzyme instead of BP Clonase II enzyme and LR Clonase II enzyme?

In the single-step protocol for the BP/LR Clonase reaction, we would not recommend substituting the BP Clonase II/LR Clonase II enzymes with BP Clonase /LR Clonase enzymes as this would result in very low recombination efficiency.

Do you have a recommended single-step protocol for BP/LR recombination?

Yes, we have come up with a single-step protocol for BP/LR Clonase reaction (http://www.thermofisher.com/us/en/home/life-science/cloning/gateway-cloning.html#1), where DNA fragments can be cloned into Destination vectors in a single step reaction, allowing you to save time and money.

How can I move my gene of interest from a Gateway-adapted expression clone to a new Destination vector as I have lost the entry clone?

We would recommend performing a BP reaction with a Donor vector in order to obtain an entry clone. This entry clone can then be used in an LR reaction with the Destination vector to obtain the new expression clone.

Can I purchase the 5X LR Clonase buffer or 5X BP Clonase buffer separately?

We do not offer the 5X LR Clonase buffer and 5X BP Clonase buffer as standalone products. They are available as part of the enzyme kits.

Do you offer Gateway vectors for expression in plants?

We do not offer any Gateway vectors for expression in plants.

Do you offer any MultiSite Gateway vectors as standalone products?

We do not offer any of the MultiSite Gateway vectors as standalone products. They are only available as part of the kits.

I obtained few or no colonies after my MultiSite Gateway LR reaction, whereas the transformation control gave colonies. Can you please offer some troubleshooting tips?

– Check whether the correct Clonase enzyme was used and whether it was functional (make sure that LR Clonase II Plus was used).
– Check whether the correct combination of Entry vectors and Destination vector was used.
– Ensure that the correct Entry clones were used (and that they were sequenced).
– Check if the recommended amount of Entry vector and Destination vector was used in the reaction.
– Perform the LR reaction positive controls to determine which Entry clone may be faulty (note that MultiSite GW 3-Fragment kit only comes with a single control vector, pMS/GW, that is used to make all control pENTR vectors).
– Check whether the correct antibiotic was used for selection.
– Check whether the att site sequences in the Destination vector are correct.
– Increase the incubation time up to 18 hours.

What is the cloning efficiency of 2-, 3-, or 4-fragment MultiSite Gateway cloning?

Standard MultiSite Gateway reactions are incubated for 16-18 hours at 25 degrees C. 2 or 4 µL (for 4 fragment recombination) aliquots are transformed into One Shot Mach1 cells. Use Mach1 cells instead of Top10 cells for the highest efficiency. For 2- and 3-fragment recombination reactions, it is important to keep optimum molar ratios of the plasmids (20 fmoles pDEST + 10 fmoles pENTR vectors). Hundreds of AmpR colonies can be obtained with a cloning efficiency of 90% (expected efficiency is a bit lower for 3-fragment recombination, usually 70-90%). For 2-fragment recombination dilute the reaction 1:10 in SOC medium and plate 50 and 100 µl. For 3-fragment recombination, plate 50 and 100 µL of each transformation. For 4 fragment recombination use 10 fmoles of each pENTR plasmid plus 20 fmoles of the pDEST vector. Incubate for 16 hours and transform into Mach1 cells. The number of colonies and transformation efficiency is normally reduced for 4 fragment recombination. The number of correct clones has been observed to be as high as 80% and as low as 30%, depending on the inserts being cloned.

What is the smallest quantity of RNA detectable by the SuperScript IV First-Strand System for RT-PCR?

Detection limits dependend on many factors, including primer design, target size, and the abundance of mRNA. The recommended template amount when working with SuperScript IV Reverse Transcriptase is 10 pg-5 µg total RNA or 10 pg-500 ng mRNA.

What size targets can be amplified by the SuperScript One-Step RT-PCR Systems?

The SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase is recommended for amplifying RNA targets up to 4.5 kb and the SuperScript IV UniPrime One-Step RT-PCR System is recommended for amplifying RNA targets up to 13 kb.

How stable are your cationic lipids that are used in transfection?

All the following lipids are stable at 4º C for at least one year:
11668-019 Lipofectamine 2000
10362-100 Cellfectin II
10459-014 DMRIE-C
10964-013 Lipofectamine PLUS
18292-011 Lipofectin
18324-012 Lipofectamine


Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

What is Gateway Cloning Technology?

Gateway Cloning Technology is an easy-to-use system for cloning and subcloning DNA segments (e.g. genes of interest), facilitating gene functional analysis, protein expression, and the integration of technology platforms. One can also readily clone PCR products into so-called Gateway "Entry" vectors. To shuttle inserts from one vector to another, the Gateway Cloning Technology uses bacteriophage lambda-based site-specific recombination. There is no need to use restriction enzymes and ligase to subclone inserts.

One advantage of Gateway Cloning Technology is that genes present in a single Gateway Entry vector can be subcloned into multiple different Gateway Destination vectors. After this 1 hour in vitro subcloning reaction, a high percentage of the colonies obtained carry the desired expression clone. For more details, please see the product manual for cat# 12535029 or cat# 12535037.