Electra Reagents Kit
The Electra Reagents Kit contains all necessary components to facilitate cloning a gene from a pMOTHER vector or a PCR product into an Electra pMOTHER or pDAUGHTER expression vector.
Learn more about the Electra Vector System.
Electra Reagents Kit Components
- Electra Buffer Mix:
- Supplied as a 10X mix, to be diluted to 1X in the final reaction mix.
- Electra Enzyme Mix:
- Supplied as a 20X mix, to be diluted to 1X in the final reaction mix. The mix contains SapI and T4 ligase, and is formulated for optimal cloning efficiency.
- Positive Control:
- A mix of pMOTHER vectors with a Tet promoter and a yellow fluorescent protein (KringleYFP) for Electra expression pDAUGHTER vectors, and a Tet promoter with a green fluorescent protein (DasherGFP) for Cas9 Electra pDAUGHTER vectors. It allows monitoring of the transfer of KringleYFP or DasherGFP into a pDAUGHTER expression vector and is seen as yellow or green colonies when plated with selection antibiotic.
|Mother Vector DNA or PCR product (20 ng final)||1|
|Daughter Vector (20 ng final)||1|
|Electra Buffer Mix* (1X)||2|
|Electra Enzyme Mix* (1X)||1|
- Combine components as listed in table above in a single 1.5ml tube. Note Daughter Vectors come pre-linearized from ATUM.
- Incubate 5 – 20 minutes at room temperature.
- Transform 2 µl of each reaction into competent cells*. Note cells used at ATUM are streptomycin resistant cell lines.
- Plate onto LB plates with selection antibiotic alone,
or with selection antibiotic and 100 µg/mL streptomycin (Teknova #L1148 Kan+strep),
or YEG plates with selection antibiotic and 16mM p-chloro-phenylalanine (Teknova #Y5700 (Kan+chloro-phe) or #Y5705 (Amp+chloro-phe)). (Protocol for making pheS or rpsL plates)
- Incubate overnight at 37°C.
To PCR your ORF: we recommend you add the following ends to your primers, as these contain the Electra sites to clone directly into Electra vectors. Add 15-20 bp of your ORF to the 3′ primer end to amplify your ORF and have it compatible with any of the Electra MOTHER or Electra DAUGHTER expression vectors.
- Forward primer with ATG start codon on primer:
5′-TACACGTACTTAGTCGCTGAAGCTCTTCTATG….(ORF beginning after ATG start codon)….
- Reverse primer:
5′-AGGTACGAACTCGATTGACGGCTCTTCTACC….(ORF Reverse Complement)….
A PCR mixture can be directly cloned into pMOTHER or pDAUGHTER vectors using the Electra reagents. However, if your PCR reaction shows multiple bands by gel analysis, it is very likely that some fragments other than your gene of interest will also contain SapI ends and may be cloned into your Electra vector. Additional screening of colonies will be useful to identify clones containing your gene of interest.
Your ORF must NOT contain any SapI recognition sites, as the Electra cloning process utilizes the type IIs enzyme SapI
Use the ATUM Bioinformatics Toolbox to facilitate primer analysis and design.
Bolt PCR Cloning Kit
ATUM has developed a simple, fast, and efficient method for cloning PCR amplified products into a plasmid vector in a 5 minute bench-top reaction. This cloning kit contains an optimized cloning mix with a linearized vector that allows cloning of any PCR product amplified with proofreading DNA polymerases (not compatible with Taq polymerase).
Bolt Cloning Kit Components
- Linearized Vector:
- Linearized pJ201 (kanR) in BKT-01 or pJ204 (ampR) vector in BKT-02 is supplied as a 10X mix, to be diluted to 1X in the final reaction mix (10X mix contains 20ng/µl linearized vector DNA and topoisomerase).
- Positive Control:
- Supplied as a PCR amplicon of KringleYFP linked to a tet promoter, 25 ng/µl, 864 base pairs, which can be cloned in either orientation.
- Sequencing Primers:
- Forward primer: 5′- TGGTAGTGTGGGGACTC-3′
- Reverse primer: 5′- TTGTCAGAATATTTAAGGGCG-3′
- PCR Primers Not Included:
- There are no sequence restrictions for Bolt cloning, so you may use any PCR primer. The Bolt vectors do not contain multiple cloning sites (MCS), so flanking restriction sites must be incorporated in your PCR primers for easy subcloning into a vector of choice.
Bolt PCR Cloning Protocol
|PCR product (~100ng) or Control||4|
|Linearized Bolt Vector (20ng) mix||1|
- Combine components as listed in table above in a single 1.5ml tube.
- Incubate 5 – 20 minutes at room temperature.
- Transform 2.5 µl of each reaction into 50 µl competent cells*. Heat shock if appropriate, and then add 500 µl SOC Broth. Shake for 1 hour at 37°C.
- Plate 100 µl on LB + selection antibiotic – pJ201 on LB agar + 30 µg/ml kanamycin; pJ204 on LB agar + 100 µg/ml ampicillin or carbenicillin.
- Incubate overnight at 37°C. Pick transformants.
A PDF of this protocol with additional vector information can be found here.
Patents: This technology is covered by issued US patents 8,323,930, and 9,102,944, and related pending applications.
TEV protease is an engineered catalytic domain of the Tobacco Etch Virus Nla cysteine protease which is used to remove fusion tags from purified proteins. ATUM offers vectors with a TEV protease recognition site (ENLYFQ/G) between the tag and the ORF. The enzyme is His-tagged and affinity purified.
- 1 mg affinity purified TEV-His protease in 50 mM Tris-HCl, pH 8, 150 mM NaCl, 40% glycerol, 1 mM DTT at 1 mg/ml
- TEV protease (100 µg/ml) shows 100% activity at 4°C overnight or 30°C for 1 hour in buffer with 50 mM Tris-HCl, pH 8, 150 mM NaCl and 1 mM DTT. While TEV protease cleaves both on and off-column, it is more efficient in solution (off-column)
- A modified 27 kDa TEV protease construct containing a 6XHis tag for easy removal using His affinity media
- TEV protease is an efficient tool for fusion protein cleavage in solution or immobilized TEV protease on streptavidin-agarose
ATUM vectors with TEV cleavage sites
|pD441-NHT||T5 promoter, N-term His, TEV cleavage site|
|pD441-PpiBT||T5 promoter, N-term PpiB, TEV cleavage site|
|pD861-NHT||Rham promoter, N-term His, TEV cleavage site|
|pD861-PpiBT||Rham promoter, N-term PpiB, TEV cleavage site|
|pD881-PpiBT||Rham promoter, N-term PpiB, TEV cleavage site, low copy|
TEV Protease Cleavage Data
TEV Protease Cleavage Process
HRV3C protease is a recombinant form of the human rhinovirus 3C cysteine protease which is used to remove fusion tags from purified proteins. ATUM offers vectors with a HRV3C protease recognition site (LEVLFQ/GP) between the tag and the ORF. The enzyme is GST-tagged and affinity purified.
- 1 mg affinity purified HRV3C-GST protease in 50 mM Tris-HCl, pH 8, 150 mM NaCl, 40% glycerol, 1 mM DTT at 1 mg/ml
- HRV3C protease at 125 ng/50 µg target protein shows 100% activity at 4°C overnight; 1 µg/50 µg target protein shows 100% activity at 4°C for 4 hours, cleavage was done in buffer with 50 mM Tris-HCl, pH 8, 100 mM NaCl, 0.5 mM EDTA, 10 mM reduced glutathione and 1 mM DTT. While HRV3C protease cleaves both on and off-column, it is more efficient in solution (off-column).
- A recombinant 47 kDa HRV3C protease construct containing a GST tag for easy removal using Glutathione (GSH) resin.
- HRV3C protease is an efficient tool for fusion protein cleavage in solution or immobilized HRV3C protease on streptavidin-agarose.
ATUM vectors with HRV3C cleavage sites
|pD441-GST||T5 promoter, N-term GST, HRV3C cleavage site|
|pD441-HMBP||T5 promoter, N-term HisMBP, HRV3C cleavage site|
HRV3C Protease Cleavage Data:
Anti-CometGFP® Polyclonal Antibody
Anti-CometGFP is a rabbit polyclonal generated by immunizing rabbits with purified CometGFP protein. The antibody was affinity purified and dialysed against PBS with 0.02% sodium azide.
- Affinity purified Rabbit IgG, lyophilized
- 200 µg of lyophilized antibody
- Add 200 µl 50% glycerol in water to make 1 mg/ml stock. Incubate on ice for 15 minutes before use
- Store at 4°C. For long term storage (> 6 months), aliquot and store at -20°C
- Positive control
- ATUM’s ProteinPaintbox® vector FPB-26-441 or FPB-26-444 with CometGFP expressed under control of the T5 promoter in E. coli can be used as a positive expression control
- Shows cross-reactivity to DasherGFP®