2. GATEWAY CLONING ?
This system invented & commercialised by
Invitrogen life technologies since the late 1990s.
It is a molecular biology method that enables
researchers to efficiently transfer DNA fragments
between plasmids using an appropriate set of
recombination sequences,the ‘Gateway att’ sites &
two proprietary enzyme mixes called as “LR clonase”
& “BP clonase”.
3. Your Application
Gene1 Gene2 Gene3 Gene4
Your Application
Gene
Protein
Localization
Gene
Gene
Protein
Purification
Gene
RNAi
Gene
Cell-Free
Gene
Protein
interaction
Gene
Gene
Entry Clone
PCR
Gene
synthesis
ORF
collection
Library
Your
Source
GATEWAY CLONING SYSTEM
Directional cloning
Maintains Reading frame
No restriction enzymes
No ligation
1hr,room temp reaction with >99%
efficiency
No resequencing
Compatible with automation
Reversible reactions
4. It enables you to access virtually any expression system in just a
few steps.
It circumvents the roadblocks of traditional restriction enzyme cloning
No need for ligase , subcloning steps or the hours spent to screen
countless colonies.
5. ADVANTAGES OF GATEWAY CLONING
Fast reactions- 1hour room temperature cloning reactions.
Accurate result- cloning reactions achieve >95%efficiency to deliver the clone you
need.
Versatile technology- easily shuttle DNA material / insert from vector to vector.
Streamlined protocol- no need for resequencing use the same clone from target
identification to validation.
6. BASICS OF GATEWAY CLONING
BP reaction- to create Invitrogen gateway entry clone.
LR reaction- to create a gateway expression clone.
One tube format- to create Gateway expression clone
from a pcr product.
Gateway vector conversion- converting your favourite
cloning vectors to gateway technology.
7. BP REACTION
• Creating a Gateway entry clone from an attB-flanked PCR product is an easy 1 hour
reaction. See below for an overview of the set-up. For more detailed information, refer
to the manual.
• Add the following components to a 1.5 ml tube at room temperature and mix:
attB-PCR product (=10 ng/µl; final amount ~15–150 ng) 1–7 µl
Donor vector (150 ng/µl) 1 µl
TE buffer, pH 8.0 to 8 µl
• Thaw on ice the Invitrogen BP Clonase II enzyme mix for about 2 minutes. Vortex the
BP Clonase II enzyme mix briefly twice (2 seconds each time).
• To each sample (Step 1, above), add 2 µl of BP Clonase II enzyme mix to the reaction
and mix well by vortexing briefly twice. Microcentrifuge briefly.
• Return BP Clonase II enzyme mix to –20°C or -80°C storage.
• Incubate reactions at 25°C for 1 hour.
• Add 1 µl of the Proteinase K solution to each sample to terminate the reaction. Vortex
briefly. Incubate samples at 37°C for 10 minutes.
8. • Transformation
• Transform 1 µl of each BP reaction into 50 µl of Invitrogen One Shot OmniMAX 2
Phage-Resistant Cells (Catalog no. C8540-03). Incubate on ice for 30 minutes.
shock cells by incubating at 42°C for 30 seconds. Add 250 µl of S.O.C. Medium
incubate at 37°C for 1 hour with shaking. Plate 20 µl and 100 µl of each
transformation onto selective plates. Note: Any competent cells with a
transformation efficiency of >1.0 × 10 8 transformants/µg may be used.
• Transform 1 µl of pUC19 DNA (10 ng/ml) into 50 µl of One Shot OmniMAX 2 T1
Phage-Resistant Cells as described above. Plate 20 µl and 100 µl on LB plates
containing 100 µg/ml kanamycin, or the appropriate selection marker for your
donor vector.
•
Expected results
An efficient BP recombination reaction will produce >1500 colonies if the entire BP
reaction is transformed and plated.
10. LR REACTION
• Transferring your gene from a Gateway entry clone to destination vector is an easy 1 hour reaction. See below for an
overview of the set-up. For more detailed information, refer to the manual.
• Add the following components to a 1.5 ml tube at room temperature and mix:
Entry clone (50-150 ng) 1–7 µl
Destination vector (150 ng/µl) 1 µl
TE buffer, pH 8.0 to 8 µl
• Thaw on ice the Invitrogen LR Clonase II enzyme mix for about 2 minutes. Vortex the LR Clonase II enzyme mix
briefly twice (2 seconds each time).
• To each sample (Step 1, above), add 2 µl of LR Clonase II enzyme mix to the reaction and mix well by vortexing
briefly twice. Microcentrifuge briefly.
• Return LR Clonase II enzyme mix to -20°C or -80°C storage.
• Incubate reactions at 25°C for 1 hour.
• Add 1 µl of the Proteinase K solution to each sample to terminate the reaction. Vortex briefly. Incubate samples at
37°C for 10 minutes.
• Transformation
Follow the protocol as indicated for the BP reaction, except use the appropriate selection marker for the LB plates
suited to your destination vector (typically 100 µg/ml ampicillin).
Expected results
An efficient LR recombination reaction will produce >5000 colonies if the entire LR reaction is transformed and
plated
12. ONE TUBE FORMAT
• If you want to transfer your attB-flanked PCR product directly into an expression clone, you can easily combine the BP and LR
reactions using the following protocol. This will potentially eliminate the transformation and DNA isolation of the Gateway entry
clone.
• In a 1.5 ml microcentrifuge tube, prepare the following 15 µl BP reaction:
attB DNA (50-100 ng) 1.0–5.0 µl
attP DNA (Invitrogen pDONR vector, 150 ng/µl) 1.3 µl
BP Clonase II enzyme mix 3.0 µl
TE Buffer, pH 8.0 add to a final volume of 15 µl
• Mix well by vortexing briefly and incubate at 25°C for 4 hours.
Note: Depending on your needs, the length of the recombination reaction can be extended up to 20 hours. An overnight
incubation typically yields 5 times more colonies than a 1 hour incubation. Longer incubation times are recommended for large
plasmids (=10 kb) and PCR products (=5 kb).
• Remove 5 µl of the reaction to a separate tube and use this aliquot to assess the efficiency of the BP reaction (see below).
• To the remaining 10 µl reaction, add:
Destination vector (150 ng/µl) 2.0 µl
LR Clonase II enzyme mix 3.0 µl
Final volume 15 µl
• Mix well by vortexing briefly and incubate at 25°C for 2 hours.
Note: Depending on your needs, the length of the recombination reaction can be extended up to 18 hours.
13. • Add 2 µl of proteinase K solution. Incubate at 37°C for 10 minutes.
1.Transform 50 µl of the appropriate competent E. coli with 1 µl of the reaction.
2.Plate on LB plates containing the appropriate antibiotic to select for expression clones.
Assessing the efficiency of the BP reaction
1.To the 5µl aliquot obtained from “One-Tube” Protocol, Step 3, above, add 0.5 µl of proteinase K solution.
Incubate at 37°C for 10 minutes.
2.Transform 50 µl of the appropriate competent E. coli with 1 µl of the reaction. Plate on LB plates
containing the appropriate antibiotic to select for entry clones.
14. GATEWAY CONVERSION
• Converting your favorite set of cloning vectors to Gateway Technology is a fairly
straightforward protocol, and will ultimately allow you to streamline your cloning and
expression process.
To convert your cloning vector to a Gateway destination vector, you will:
• Choose the appropriate reading frame cassette to use depending on your needs.
• Linearize the vector you wish to convert with a restriction enzyme of choice. If you use a
restriction enzyme that generates an overhang, you will need to blunt the ends.
• Remove the 5' phosphates from the vector using calf intestinal alkaline phosphatase.
• Ligate the reading frame cassette into your vector using T4 DNA ligase.
• Transform the ligation reaction into One Shot ccdB Survival Competent E. coli and select for
transformants.
• Analyze transformants.
15. GATEWAY SYSTEM relies
on five sets of specific
and non cross reacting
att sequences.
The specificity is given
by the 7 nucleotides of
the core region.
16. GATEWAY RECOMBINATION CLONING
VS TRADITIONAL RESTRICTION ENZYME
CLONING
Steps GATEWAY CLONING RESTRICTION ENZYME
CLONING
Existing primers ? Yes No
Vector ready for cloning ? Yes No
Ligation reagents included ? Yes No
Competent cells separately ? Included Purchase separately: 0 hours
Prepare: upto 6hours
Vector clean up ? No Yes
PCR FRAGMENT cleanup ? No Yes
Recombination efficiency ? upto 95% ~50%
17. GLOSSARY
att site- A defined length of DNA that constitutes a recombination site.There are 4 classes of
att sites called attB,attP,attL,attR.
ccdB gene- A counterselectable gene that allows for negative selection of unwanted
byproduct plasmid after recombination.
Entry(pENTR)clone- A vector that contains your gene of interest flanked by attL or attR
sites.
Donor(pDONR)vector- Avector with attP sites flanking a counterselectable gene that
recombines with a gene of interest flanked by attB sites.
Destination(DEST)vector- An application geared vector with attR sites flanking a
counterselectable gene that will recombine with one or more entry clones.
Multisite Gateway Technology- A system that allows simultaneous assembly of multiple
DNA fragments into a single destination vector.
