Cloning – Classic LR-Reaction II

by A. Untergasser (contact address and download at www.untergasser.de/lab)
Version: 1.0 - Print Version (.PDF)

ATTENTION: This is expensive. One reaction as described here is 5 Euro!

  1. Make sure you have one ENTR and one DEST clone for "classic" Gateway
  2. Measure the DNA concentration of both constructs
  3. Calculate the volume in µl needed of ENTR plasmid (25 ng)
    µl needed = 25 ng needed / (concentration in ng/µl)
  4. Calculate the volume in µl needed of DEST plasmid (50 ng)
    µl needed = 50 ng needed / (concentration in ng/µl)
  5. Prepare in a new eppi the Gateway reaction
    ENTRTM-vector ( 25 ng) 1µl
    DESTTM-vector (50 ng) 1µl
    add water to a total volume of 4 µl 1µl
  6. Remove the 5 x LR-Clonase II Enzyme Mix from -20°C and vortex 2 x 2 sec
    This is expensive stuff don't leave it to rot in the ice-bucket!
  7. Add 1 µl of the 5 x LR-Clonase Enzyme II Mix to the Gateway reaction and mix well
  8. Store the enzyme mix and buffer immediately at -80°C !!!
  9. Incubate at room temperature for 1 hour
  10. Add 0,5 µl of Proteinase K solution and incubate for 10 min at 37°C
  11. Transform bacteria
    For electro competent cells use 2 µl, for chemical competent all.
  12. Plate bacteria with proper antibiotic selection

Materials needed:

Gateway® Enzyme: LR-Clonase TM (# 11791-020) by Invitrogen

Commented Protocol:

1. Make sure you have one ENTR and one DEST clone for "classic" Gateway

You need one ENTRTM clone with the "classical" combination attL1 and attL2 and the DESTTM vector MUST have attR1 and attR2 sites, or it will not work.

2. Measure the DNA concentration of both constructs

The amount of ENTRTM is not so important as in a multiple Gateway® reactions, because it is more efficient. If you want to optimize you can calculate equimolar amounts of both plasmids as described in the multiple Gateway® protocol. Here we use double the amount of DESTTM-vector, because most of the ones we use are round and about double the size of the ENTRTM clones.

3. Calculate the volume in µl needed of ENTR plasmid (25 ng)

µl needed = 25 ng needed / (concentration in ng/µl)

Dilute in such way that 1 µl contains 25 ng  - this should be easy.

4. Calculate the volume in µl needed of DEST plasmid (50 ng)

µl needed = 50 ng needed / (concentration in ng/µl)

Dilute in such way that 1 µl contains 50 ng - this should be easy.
The DESTTM-vector should be tested for low background colonies (due to a mutated ccdB-gene) when transferred in DH5alpha-bacteria.

5. Prepare in a new eppi the Gateway reaction

ENTRTM-vector ( 25 ng) 1µl
DESTTM-vector (50 ng) 1µl
add water to a total volume of 4 µl 1µl

6. Remove the 5 x LR-Clonase II Enzyme Mix from -20°C and vortex 2 x 2 sec

This is expensive stuff don't leave it to rot in the ice-bucket!

7. Add 1 µl of the 5 x LR-Clonase Enzyme II Mix to the Gateway reaction and mix well

It is most efficiently mixed by pipetting up and down, do not vortex.

8. Store the enzyme mix and buffer immediately at -80°C !!!

This is expensive stuff don't leave it to rot in the ice-bucket!

9. Incubate at room temperature for 1 hour

10. Add 0,5 µl of Proteinase K solution and incubate for 10 min at 37°C

This step will enhance the reaction ca. 2fold.

11. Transform bacteria

For electro competent cells use 2 µl, for chemical competent all.

Electroposation requires low concentrations of salts, that's why we can not use too much.

12. Plate bacteria with proper antibiotic selection

Plate 1/10 on one plate and the rest on another plate. Sometimes you get so many clones that it could be difficult to pick one.
All our Binary-vectors are spectinomycin resistant. If you are not sure, check first.

Known Issues:

References and Comments:

I developed this protocol myself, because the supplied instructions were not clear enough and complex. I did it as described before many times and never had any problems.

Gateway®, TOPO®, pENTR TM, pDONRTM, pDEST TM BP-ClonaseTM and LR-ClonaseTM are protected trademarks of Invitrogen.
Please visit Invitrogen for further information and for the acquisition of the needed materials.

How to cite this page in publications:

This document can be cited like this:

Untergasser A. “Cloning – Classic LR-Reaction II” Untergasser's Lab. Summer 2006. (include here the date when you accessed these page).
<http://www.untergasser.de/lab/protocols/lr_classic_gateway_reaction_ii_v1_0.htm>.

Please Do Not Reprint This Article:

This article is copyrighted. Please do not reproduce this article in whole or part, in any form, without obtaining my written permission.