Reagents

Enzymes, kits and antibodies to speed up your research.

Reagents Catalog:

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.

Add Electra Reagents Kit to cart

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.
Electra Vector System Process diagram

Electra Protocol

Component Volume (µl)
Total Volume 20
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
Sterile ddH2O 15
*From the Electra Cloning Kit
  1. Combine components as listed in table above in a single 1.5ml tube. Note Daughter Vectors come pre-linearized from ATUM.
  2. Incubate 5 – 20 minutes at room temperature.
  3. Transform 2 µl of each reaction into competent cells*. Note cells used at ATUM are streptomycin resistant cell lines.
  4. 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)
  5. Incubate overnight at 37°C.
ATUM has tested the Electra vectors in DH10B E. coli cells. We recommend the use of competent DH10B cells for all Electra transformations.

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)….

Additional Info

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.

A PDF of this protocol can be found here.PDF image
A PDF to make PheS or rpsL plates can be found here.PDF image

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).

  Add BKT-01 (KanR, Bolt Cloning Kit) to cart


  Add BKT-02 (ampR, Bolt Cloning Kit) to cart

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

Component Volume (µl)
Total Volume 10
PCR product (~100ng) or Control 4
Linearized Bolt Vector (20ng) mix 1
Sterile ddH2O 5
  1. Combine components as listed in table above in a single 1.5ml tube.
  2. Incubate 5 – 20 minutes at room temperature.
  3. 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.
  4. 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.
  5. Incubate overnight at 37°C. Pick transformants.
ATUM has tested the Electra vectors in DH10B E. coli cells. We recommend the use of competent DH10B cells for all Electra transformations.

A PDF of this protocol with additional vector information can be found here.PDF image

Patents: This technology is covered by issued US patents 8,323,930, and 9,102,944, and related pending applications.

TEV protease

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.

Amount
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
Activity
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)
Properties
A modified 27 kDa TEV protease construct containing a 6XHis tag for easy removal using His affinity media
Applications
TEV protease is an efficient tool for fusion protein cleavage in solution or immobilized TEV protease on streptavidin-agarose
Add TEV protease (ENZ-01) to cart

ATUM vectors with TEV cleavage sites

Catalog # Feature
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

Reagents - TEV Protease Gel Image

Figure: TEV protease cleaves at the TEV recognition site to efficiently remove the 6XHis Tag. Purification of KringleYFP and removal of 6XHis tag from the His-KringleYFP and TEV-His protease following cleavage with TEV-His protease is shown. KringleYFP with N-terminal 6XHis and TEV cleavage site ~28kD band (shown by red arrow) was cleaved with 100 µg/ml TEV protease at 4°C O/N or at 30°C for 1 hour. Commercially available TEV-His-GST protease was used for comparison; ‘no enzyme’ samples were run as negative controls. A SDS-PAGE gel was run with total (load), flow through and eluted fractions from an IMAC column and Coomasie stained. Total fraction shows KringleYFP and the TEV protease in samples treated with TEV protease (bands shown by black and blue arrows); flow through fractions showed only the His tag cleaved KringleYFP, seen as ~ 26kD band (shown by blue arrows) in TEV protease treated samples; no cleavage of KringleYFP-His was seen in the ‘no enzyme’ control. TEV protease cleaves efficiently at 4°C or 30°C. Although KringleYFP is stable at 30°C for fast TEV protease cleavage, for other proteins we recommend testing stability of your protein before incubation at 30°C.

TEV Protease Cleavage Process

Reagents - TEV Protease Cleavage Process

TEV Protease Cleavage Process. A general schematic for cleavage of the 6X His tag with TEV protease and purification is shown. KringleYFP was expressed with a N-terminal TEV protease cleavage site and a 6XHis tag (His-TEV_PCS-YFP). The 6XHis tag was cleaved in solution after the first IMAC (immobilized metal ion chromatography) column purification, dialysed into buffer (50 mM Tris-HCL, pH 8, 150 mM NaCl, 1 mM DTT) to remove imidazole and cut with 100 µg/ml TEV-His protease with mixing at 4°C overnight and at 30°C for 1hr. Samples were re-run on a IMAC column to remove His-tagged enzyme TEV-protease and the cleaved 6XHis tag from KringleYFP. The flow through has the purified tag-free protein.


HRV3C Protease

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.

Amount
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
Activity
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).
Properties
A recombinant 47 kDa HRV3C protease construct containing a GST tag for easy removal using Glutathione (GSH) resin.
Applications
HRV3C protease is an efficient tool for fusion protein cleavage in solution or immobilized HRV3C protease on streptavidin-agarose.
Add HRV3C protease (ENZ-02) to cart

ATUM vectors with HRV3C cleavage sites

Catalog # Feature
pD441-GST T5 promoter, N-term GST, HRV3C cleavage site
pD441-HMBP T5 promoter, N-term HisMBP, HRV3C cleavage site

HRV3C Protease Cleavage Data:

Reagents - HRV3C Cleavage Activity

Cleavage of GST tag with HRV3C protease. The target protein, a RNA ligase with a HRV3C protease cleavage site (LEVLFQ/GP_PCS) and a GST tag, was purified on a Glutathione matrix. The eluted protein was incubated at 4°C for 4 hours and overnight with HRV3C-GST protease at various enzyme dilutions to determine the optimal enzyme concentration required to cleave the target. At 4 hours, a 1:100 ratio of enzyme to target protein (0.5 µg protease to 50 µg target protein) showed complete cleavage of the GST tag from the target. Complete cleavage was observed even at a 1:400 ratio of protease to target protein (125 ng protease to 50 µg of target protein).

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.

Type
Affinity purified Rabbit IgG, lyophilized
Amount
200 µg of lyophilized antibody
Reconstitution
Add 200 µl 50% glycerol in water to make 1 mg/ml stock. Incubate on ice for 15 minutes before use
Storage
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
Specificity
Shows cross-reactivity to DasherGFP®
Add Anti-CometGFP (AB-02) to cart