PCR PRODUCT CLONING AND SEQUENCING |
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1. | PCR-TRAP Cloning System - for cloning PCR products |
| For positive-selection cloning of PCR products through blunt-end ligation. Allows direct cloning of PCR products without purification.
This system uses a third generation cloning vector that features positive selection for cloning PCR products. Only recombinant plasmids confer antibiotic resistance, making PCR-TRAP extremely efficient. There is no need for any post-PCR manipulation before cloning, since a significant fraction of PCR products do not contain the 3' overhanging A (Clark, 1988, Nucleic Acids Res. 16:9677) The mechanism of this unique cloning system involves the phage Lambda repressor gene, cI, which has been incorporated into the PCR-TRAP Vector. When transcribed, the gene codes for a repressor protein which binds to the Lambda right operators Or1 to Or3 of the cro gene, turning off the promoter which drives the TetR gene on the plasmid. Therefore, cloning the PCR product directly into the cI gene leads to the inactivation of the repressor gene and thus the expression of the TetR gene. So the PCR-TRAP Cloning System is indeed a cloning TRAP! request info | |
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2. | PCR-TRAP Vector Primer Kit - for cloning PCR products |
| This kit contains some of the essentail components of the PCR-TRAP Cloning System. Other components such as T4 DNA ligase, Component cells, and Tetracycline are not provided. request info | |
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3. | Riboprobe Primer Set - for creating Riboprobe templates |
| For PCR amplification of cDNAs cloned into the PCR-TRAP® system for riboprobe preparation.
This reaction creates a template for riboprobe synthesis by placing the T7 and SP6 promoter sites adjacent to the insert. request info | |
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4. | ReversePrime™ cDNA Labeling Kit |
| For labeling cDNA probes for use in "Reverse Northern" blots.
ReversePrime Kit provides a complete system for labeling cDNA for use in "Reverse Northern" blots to conduct differential screening of positive clones generated by differential display. This kit is ideal for either dot blot or colony hybridization screening of differential display PCR products cloned into the PCR-TRAP® Cloning System. request info | |
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5. | HotPrime™ DNA Labeling Kit |
| For optimized labeling of differential display fragments with radioactively labeled dATP.
The HotPrime DNA labeling kit features three unique improvements over the traditional "random priming" method which allow for radioactive labeling of DNA probes with at least 5-10X more specific radioactivity. It is designed to efficiently label cDNA probes isolated from differential display for Northern or Southern blot analysis as well as library screening. However, the HotPrime Kit can also label DNA probes for any other application to a similar high specific activity. request info | |
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6. | AidSeq™ Primer Sets - for sequencing DNA products |
| AidSeq™ Primer Set A
Primer pair for sequencing DNA cloned into vectors such as TA cloning vector (Invitrogen) and pGEM (Promega) that contain SP6 and T7 promoter binding sites. request info Cat # | Quantity | P201 | 50 reactions |
AidSeq™ Primer Set B
Primer pairs for Sequencing DNA cloned into M13 and pUC related vectors such as pBlueScript (Stratagene). request info Cat # | Quantity | P202 | 50 reactions |
AidSeq™ Primer Set C
Primer pairs for Sequencing DNA clined into PCR-TRAP® cloning vector. request info Cat # | Quantity | P203 | 50 reactions |
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1. | AP-TAG CLONING SYSTEMS |
| For non-radioactive detection of receptor/ligand interaction.
AP-TAG Kit A
This is the second generation of AP-TAG technology. A secreted ligand or soluble receptor can be fused with secreted alkaline phosphatase (AP) at either its N- or C-terminus to produce an "AP-body". The resulting AP fusion protein can be expressed as a secreted protein and used directly as highly sensitive affinity agents much like an antibody. request info | Cat # | Quantity | AP-TAG Kit A - Academic
AP-TAG Kit A - Industry | Q201
Q201P | Kit
Kit |
AP-TAG Kit B
This single vector system is the third generation AP-TAG technology. A secreted ligand or soluble receptor can be fused with secreted alkaline phosphatase (AP) at either its N- or C-terminus to produce an "AP-body". The resulting AP fusion protein can be expressed as a secreted protein and used directly as highly sensitive affinity agents much like an antibody. The epitope tags (6xHis and myc) allow easy purification, detection and interaction assays (IP) of the AP-fusion proteins. request info | Cat # | Quantity | AP-TAG Kit B - Academic
AP-TAG Kit B- Industry | Q202
Q202P | Kit
Kit |
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2. | SUPER-COMPETENT CELLS |
| For Transformation of AP-TAG Cloning Vectors.
GH2/P3 Supercompetent Cells
For transformation of pAPtag-2 and pAPtag-4 vectors.
The pAPtag-2 and pAPtag-4 AP-fusion cloning vectors contain the supF gene which confers both ampicillin and tetracycline resistance when transformed into the GH2/P3 Supercompetent Cells. pAPtag-2 and pAPtag-4 vectors will not confer antibiotic resistance in an E. coli host which does not contain the P3 episome. request info AT Antibiotics Mix
For selection of pAPtag-2 and pAPtag-4 plasmids.
Each tube of AT antibiotics mix is conveniently packaged to prepare ampicillin (25 mg/mL) and tetracycline (10 mg/mL) plates for 1 L of LB-agar. request info | Cat # | Quantity | AT Antibiotics Mix (1000X) | Q601 | 1 mL |
GH Competent Cells
For transformation of pAPtag-5 vector
The pAPtag-5 AP fusion cloning vector contains the ampicillin resistance gene. It can be easily and efficiently transformed and propagated in GH Competent cells. request info Cat # | Quantity | L301 | 6 x 0.5 mL |
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3. | ANTIBODIES AGAINST ALKALINE PHOSPHATASE |
| AP Western Blot Kit
For immunoblotting of AP fusion proteins.
This kit contains the AP Antibody (rabbit polyclonal) which is specific to human placental secreted AP as well as two controls for AP Western blots. request info AP Antibody (human placenta) - Monoclonal
For ELISA Assay of Alkaline Phosphatase or IP (Immunoprecipitation).
This Monoclonal AP Antibody (human placental) is purified IgG 2a with a concentration of approximay 2.3 mg/mL. It is purified by DEAE chromatography and is in 15mM potassium phosphate buffer, 150mM sodium chloride, 0.1% sodium azide, pH 7.2. request info Cat # | Quantity | Q320
Q321 | 100 µg
1 mg |
Monoclonal AP Antibody-Sepharose Beads
For one-step purification of AP-fusion proteins and proteins interacting with AP-fusion proteins. request info Cat # | Quantity | Q330
Q331
Q332 | 100 µL
500 µL
1 mL |
Antigen Elution Solution
For eluting AP fusion protein from the Monoclonal AP Antibody-Sepharose Beads.
This specially formulated elution buffer can break the extremely tight interaction between the antibody-antigen, allowing up to 60% recovery of the bound antigen. request info Cat # | Quantity | Q340
Q341 | 10 mL
50 mL |
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4. | CELL LINES FOR AP-TAG VECTOR TRANFECTION AND AP-BODY SECRETION |
| 293T Cells
For transfection with pAPtag vectors.
293T is a human embryonic kidney cell line commonly used for transfection assays. Due to the expression of the large T antigen in the cell, plasmids with SV40 origin of replication (such as pAPtag-2, pAPtag-4, and pAPtag-5) can be transiently transfected and give extremely high levels of expression of AP fusion proteins (e.g. ligand-AP fusion proteins). request info Cat # | Size | Q401 | 5 x 106 Cells/Vial |
293T/pAPtag-4 Stable Cell Line
For production of high levels of AP alone.
The 293T/pAPtag-4 stable cell line is used to produce high levels of secreted human placental alkaline phosphatase (AP) which can be used as a negative control for a ligand-AP or soluble receptor-AP fusion protein in cell surface binding assays or cell staining. request info Cat # | Size | Q402 | 5 x 106 Cells/Vial |
Co-transfection Vectors
For use as a selectable marker for transfection of cultured mammalian cells.
The pSV-Hygro vector confers hygromycin resistance and the pBabe-Puro vector confers puromycin resistance when co-transfected into cells. request info | Selectable Marker | Cat # | Quantity | pSV-Hygro co-transfection vector
pBabe-Puro co-transfection vector | Hygromycin
Puromycin | Q455
Q456 | 10 µg
10 µg |
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5. | RECEPTOR/LIGAND EXPRESSION CLONING |
| pMT21-Neo Mammalian Expression Cloning Vector
For construction of expression cDNA libraries.
This cloning vector has been used extensively to construct mammalian expression cDNA libraries. The vector contains an SV40 origin of replication and the major adeno late promoter (PMAL) in front of Neo resistance cDNA insert flanked by an EcoR I and Xho I site. Using the Stratagene cDNA Synthesis Kits generally results in cDNA ends with EcoR I and Xho I sites, which can be directionally cloned into the pMT21-NeoR vector. request info Expression Cloning Kit
For expression cloning of cell surface receptor/ligand using AP fusion proteins (AP-bodies).
This kit consists of a cos-1 host cell line (commonly used for expression cloning by panning) and a positive control receptor/ligand-AP pair. request info | |
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6. | AP ACTIVITY ASSAY REAGENTS AND BUFFERS |
| AP Assay Reagent A
For AP Activity Assay.
The AP assay reagent A is formulated specifically for measuring the enzymatic activity of the alkaline phosphatase (AP). AP Assay Reagent S
For Cell Staining.
The AP Assay Reagent S is formulated specifically for cell staining or Western blotting analysis of enzymatic activity of the alkaline phosphatase (AP). HBHA Wash Buffer
For Receptor Binding Assay.
HBHA wash buffer consists of Hank's balanced salt solution with 0.5 mg/mL BSA and 20 mM HEPES, pH 7.0. This buffer has been used extensively for ligand-receptor binding assays. Cat # | Quantity | Q503S
Q503L | 100 mL
500 mL |
Cell Lysis Buffer
For Cell Lysis in Receptor Binding Assay .
The cell lysis buffer is used in ligand-receptor binding assay. This buffer allows rapid lysis of the cells and removal of cell nuclei before bound AP activity is measured. | |
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