FX cloning System
(Kit #
1000000039
)
Depositing Labs: Raimund Dutzler, Eric Geertsma
The 35 plasmid FX cloning kit can be used to insert a sequence of interest into a cloning vector and easily swap into expression vectors for several organisms with a variety of N- and C-terminal tags.
This kit consists of one 96-well plate, and will be shipped as bacterial glycerol stocks on dry ice. Samples should be frozen at -80°C immediately upon arrival. Individual plasmids can be ordered from each plasmid page and will be shipped as bacterial stabs.
Original Publication
A versatile and efficient high-throughput cloning tool for structural biology. Geertsma ER and Dutzler R. Biochemistry 2011 Apr 19;50(15):3272-8. doi: 10.1021/bi200178z. Epub 2011 Mar 25. PubMed PMID 21410291.
Description
The FX cloning system (fragment exchange cloning) is an efficient, inexpensive cloning method that utilizes a Type IIS restriction enzyme and negative selection markers. This system is suitable for both prokaryotic and eukaryotic expression systems and combines the advantages of established recombination- and ligation-independent cloning methods, while reducing undesired cloning-related sequences added in the process.
The FX cloning system uses a set of three initial cloning vectors (pINIT_cat, pINIT_kan and pINIT_tet) for the initial cloning of a PCR product into one of the vectors, which differ by bacterial resistances. The subcloning of the ORF from the pINITIAL vector into an expression vector utilizes the same cloning procedure. PCR products can also be directly cloned into expression vectors.
The FX cloning Kit contains 3 initial cloning vectors, 12 E. coli expression vectors, 3 L. lactis expression vectors, 4 P. pastoris expression vectors, 4 S. cerevisiae expression vectors, 4 mammalian expression vectors, 2 Xenopus oocyte expression vectors and 3 insect expression vectors. A variety of expression vectors are available containing several combinations of 3C protease-cleavable N-terminal and C-terminal tags , including decaHis, Myc, Streptavidin Binding Peptide and Avi tags plus GFP.
FX cloning Method Overview
As described in the original publication, a PCR product containing a sequence of interest can be cloned into either an FX sequencing vector or an FX expression vector.
FX Subcloning Overview
Once a sequence of interest has been cloned into an FX sequencing vector, it can easily be transferred into an expression vector by digestion and ligation.
Kit Documentation
When receiving kit as frozen glycerol stocks on dry ice:
FX cloning kit 262.3Â KBResources
More information on these plasmids and their use can be found in the original publication.
The FX Cloning website has an automated primer design function to assist with the FX Cloning method.
How to Cite this Kit
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.
For your Materials and Methods section:
"The plasmid kit used for fragment exchange cloning was a gift from Raimund Dutzler and Eric Geertsma (Addgene kit # 1000000039)"
For your Reference section:
A versatile and efficient high-throughput cloning tool for structural biology. Geertsma ER and Dutzler R. Biochemistry 2011 Apr 19;50(15):3272-8. doi: 10.1021/bi200178z. Epub 2011 Mar 25. PubMed PMID 21410291.
FX cloning Kit - #1000000039
- Resistance Color Key
Each circle corresponds to a specific antibiotic resistance in the kit plate map wells.
- Inventory
Searchable and sortable table of all plasmids in kit. The Well column lists the plasmid well location in its plate. The Plasmid column links to a plasmid's individual web page.
- Kit Plate Map
96-well plate map for plasmid layout. Hovering over a well reveals the plasmid name, while clicking on a well opens the plasmid page.
Resistance Color Key
| Chloramphenicol and Ampicillin | |
| Chloramphenicol and Bleocin (Zeocin) | |
| Chloramphenicol and Kanamycin | |
| Tetracycline | |
| Chloramphenicol |
Inventory
| Well | Plasmid | Resistance |
|---|---|---|
| A / 1 | pBXNH3 | Chloramphenicol and Ampicillin |
| A / 2 | pBXC3H | Chloramphenicol and Ampicillin |
| A / 3 | pBXNH3CA | Chloramphenicol and Ampicillin |
| A / 4 | pBXC3GH | Chloramphenicol and Ampicillin |
| A / 5 | pBXCA3GH | Chloramphenicol and Ampicillin |
| A / 6 | pBXCA3H | Chloramphenicol and Ampicillin |
| A / 7 | pBXNAC3GH | Chloramphenicol and Ampicillin |
| A / 8 | pBXNAC3H | Chloramphenicol and Ampicillin |
| A / 9 | pBXNH3A | Chloramphenicol and Ampicillin |
| A / 10 | pREXC3GH | Chloramphenicol and Ampicillin |
| A / 11 | pREXC3H | Chloramphenicol and Ampicillin |
| A / 12 | pREXNH3 | Chloramphenicol and Ampicillin |
| B / 1 | pYEXNH3 | Chloramphenicol and Ampicillin |
| B / 2 | pYEXNHG3 | Chloramphenicol and Ampicillin |
| B / 3 | pYEXC3H | Chloramphenicol and Ampicillin |
| B / 4 | pYEXC3GH | Chloramphenicol and Ampicillin |
| B / 5 | pcDXNSM3 | Chloramphenicol and Ampicillin |
| B / 6 | pcDXNSMG3 | Chloramphenicol and Ampicillin |
| B / 7 | pcDXC3MS | Chloramphenicol and Ampicillin |
| B / 8 | pcDXC3GMS | Chloramphenicol and Ampicillin |
| B / 9 | pTLNX | Chloramphenicol and Ampicillin |
| B / 10 | pTLNXA7 | Chloramphenicol and Ampicillin |
| B / 11 | pFBXNH3 | Chloramphenicol and Ampicillin |
| B / 12 | pFBXC3H | Chloramphenicol and Ampicillin |
| C / 1 | pFBXC3GH | Chloramphenicol and Ampicillin |
| C / 2 | pICXNH3 | Chloramphenicol and Bleocin (Zeocin) |
| C / 3 | pICXNHG3 | Chloramphenicol and Bleocin (Zeocin) |
| C / 4 | pICXC3H | Chloramphenicol and Bleocin (Zeocin) |
| C / 5 | pICXC3GH | Chloramphenicol and Bleocin (Zeocin) |
| C / 6 | p7XNH3 | Chloramphenicol and Kanamycin |
| C / 7 | p7XC3H | Chloramphenicol and Kanamycin |
| C / 8 | p7XC3GH | Chloramphenicol and Kanamycin |
| C / 9 | pINIT_kan | Chloramphenicol and Kanamycin |
| C / 10 | pINIT_tet | Tetracycline |
| C / 11 | pINIT_cat | Chloramphenicol |