- PCR your ORF of interest using the following 5'sequence added onto the primers
- forward primer (at start) 5' GGGGACAAGTTTGTACAAAAAAGCAGGCTaaXXXX
- two extra bp (aa) added
- XXXX=your gene sequence (usually ATGXXXXX)
- of potential note: In eukaryotes, the preferred sequence context is --GCC ACC ATG G-- around the initiating methionine, with the A at -3 being most important, and a purine at +4 (where the A of the ATG is +1), preferably a guanine (G), being next most influential (9). Having an A at -3 is enough to make most ribosomes choose the first AUG of an mRNA in plants, insects, yeast, and mammals (known as a Kozak sequence). So it might be smart to add 3 bp upstream of start when cloning (ANNATGxxx)
- reverse primer (at end) 5' GGGGACCACTTTGTACAAGAAAGCTGGGTtZZZZ
- one extra base added (t)
- ZZZZ=your gene sequnce usually stop codon onward (e.g., TTAZZZZ) don't forget to reverse complement...
- If you're gonna use a C-term tag, omit the stop codon and make sure it's in frame (should be as written w/ 1 extra base, but double check)
perform PCR Edit
- use a high fidelity pol (e.g., Phusion)
Water to 20ul
5X Phusion HF Buffer 4 µl
10 mM dNTPs 0.4 µl
10 µM Forward Primer 1 µl (note stock primers are usually 100uM so dilute 1/10 first)
10 µM Reverse Primer 1 µl
Template DNA variable < 250 ng
DMSO to 3% (optional) (if high GC content)
Phusion Pol 0.2 µl
Phusion pol manual notes:
We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C). All components should be mixed and centrifuged prior to use. It is important to add Phusion DNA Polymerase last, in order to prevent any primer degradation caused by the 3´→5´ exonuclease activity. Phusion DNA Polymerase may be diluted in 1X HF or GC Buffer just prior to use in order to reduce pipetting errors. Please note that protocols with Phusion DNA Polymerase may differ from protocols with other standard polymerases. As such, conditions recommended below should be used for optimal performance.
BP or LR reaction (adapted from Alberti et al 2007 Yeast)Edit
- 15-150 ng expression clone (BP) or entry clone (LR): 1 µl.
- ~50-150 ng entry vector (BP) or destination vector (LR): 1 µl.
- 1X TE (pH 8): 2 µl.
- BP (or LR) Clonase II mix (Invitrogen): 1 µl.
- Incubate at room temperature for 1 h (ON 4C maybe higher efficieny, but 1hr RT works well for me). Transform 2–5 µl of the reaction into DH5α, TOP10 or other ccdB-sensitive cells. Select for transformants on LB agar plates containing Kanamycin for pDONR221 (or LB + 100 µg/ml Amp for other).
- include controls for transformation (entry vector of destination vector alone - this will show you the frequency of ccdB mutations as opposed to successful BP/LR rxn. So ideally your rxns will have more colonies than vector only.)
“One-Tube” Protocol for Cloning attB-PCR Products Directly into Destination VectorsEdit
1. 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 (pDONR™vector, 150 ng/μL) 1.3 μL
Gateway BP Clonase II enzyme mix 3.0 μL
TE Buffer, pH 8.0 add to a final volume of 15 μL
2. 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).
3. Remove 5 μL of the reaction to a separate tube and use this aliquot to assess the efficiency of the BP reaction (see below).
4. To the remaining 10 μL reaction, add: Destination vector (150 ng/μL) 2.0 μL Gateway LR Clonase II enzyme mix 3.0 μL Final volume 15 μL
5. 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.
6. Add 2 μL of proteinase K solution. Incubate at 37°C for 10 minutes.
7. 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 expression clones.
Assessing the Efficiency of the BP Reaction 1. To the 5 μL 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.
addgene yeast gateway plate mapEdit
Note: To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip, puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate. To patch the hole, use sterile tape or a portion of a fresh aluminum seal.