Feb 07, 2017
Version 2
Gateway BP recombination of attB tailed PCR products into pDONR/zeo (5 µl assay) V.2
External link: https://www.thermofisher.com/order/catalog/product/11789020
Protocol Citation: Johannes Wolfram JWD Debler: Gateway BP recombination of attB tailed PCR products into pDONR/zeo (5 µl assay). protocols.io https://dx.doi.org/10.17504/protocols.io.g5rby56
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: February 07, 2017
Last Modified: March 27, 2018
Protocol Integer ID: 5009
Abstract
This is a slightly modified version of Thermo Fisher's gateway BP protocol which uses less enzyme and is therefore more economical.
Calculate amount of attB PCR product needed
Calculate amount of attB PCR product needed
ng of attB PCR product = size of attB tailed PCR product in bp * 0.0165
eg. 778 bp * 0.0165 = 12.9 ng
If your purified PCR product has a concentration of 25 ng/ul you would use 0.5 µl in the next step.
Note
Your attB PCR product should be purified either by gel purification or with a PCR clean-up kit
Combine in 0.2 ml PCR tube
Combine in 0.2 ml PCR tube
| attB PCR product | x µl (as calculated in step 1) | |
| pDONR/zeo (150 ng/µl) | 0.5 µl | |
| TE buffer pH 8.0 | to a total volume of 4 µl |
BP Clonase II enzyme mix
BP Clonase II enzyme mix
Remove from freezer and vortext for 2 seconds.
add BP Clonase II
add BP Clonase II
Add 0.5 µl of BP Clonase II enzyme mix and mix well.
Incubate at room temperature
Incubate at room temperature
Incubate for 1 hour at room temperature (2-3 hours or overnight for more colonies).
01:00:00
Stop reaction with Proteinase K
Stop reaction with Proteinase K
Add 0.5 µl Proteinase K, mix well and incubate for 10 min at 37°C.
00:10:00
Transform into Omnimax 2 E.coli
Transform into Omnimax 2 E.coli
Combine 1 µl BP product with 50 µl Omnimax 2 competent E.coli cells in 1.5 ml tube.
Incubate on ice
Incubate on ice
00:10:00
Heat shock
Heat shock
30 seconds at 42°C
Reco on ice
Reco on ice
00:02:00
Add SOC
Add SOC
Add 500 µl SOC.
Incubate
Incubate
1 hour at 37° C at 250 rpm on shaking incubator.
01:00:00
Plate out
Plate out
Plate 20 ul (or more) on LB plates containing 50 ug/ml zeocin and incubate at 37° C over night.
Note
I have observed that the older your BP Clonase II enzyme mix is, the more of the BP reaction you need to plate out. You can therefore concentrate your cells by centrifuging at 1000 x g for a couple of minutes, decant most of the supernatant and resuspend the cell pellet in about 50-100 µl of the leftover supernatant. Then plate all of that on the LB + zeocin plate.