{"product_id":"neb-e8101s","title":"New England Biolabs, E8101S, Ph.D.™ Peptide Display Cloning System","description":"The Ph.D.™ Peptide Display Cloning System contains M13KE vector, the parent vector of NEB’s Ph.D. libraries. The included M13 extension primer is used to make DNA duplex out of a user provided synthetic oligo, with or without random bases. The display system is best used to display small peptides, i.e. sequences of 50 amino acids or less.  \u003cb\u003eRelated Categories\u003c\/b\u003e Phage Display  \u003cb\u003eApplications\u003c\/b\u003e Phage Display  \u003cb\u003eFAQ\u003c\/b\u003e Q: Can cDNA libraries be cloned into M13KE? A: In general, M13 is not amenable to cDNA expression, due to the requirement for in-frame expression between the leader sequence (required for secretion) and the N-terminus of coat protein pIII or pVIII. The consequence of this requirement is that an insert must be in the correct reading frame at both ends (p = 1\/9) and contain no in-frame stop codons (p = [61\/64] n\/3, where n is the average insert length in base pairs) in order for the corresponding protein sequence to be properly fused to the coat protein. This results in a vanishingly small number of productive clones in M13 cDNA libraries. In contrast, expression of cDNA inserts as C-terminal coat protein fusions is possible in the phage T7 display systems. Q: Can I display antibody fragments or proteins at the N-terminus of pIII coat protein with M13KE? A: In the Ph.D.™ phage display system each copy of pIII displays the encoded peptide sequence. For this reason any insert that interferes with phage particle assembly or pIII function will not make viable phage. Typically phagemid systems, where a second copy of native coat protein is provided on by helper phage, are used to display protein-sized inserts on M13. We consider 25-50 amino acids to be the maximum range for this system. To some extent it is also sequence and secondary structure dependent. Q: What are the differences between pIII and pVIII display? A: Filamentous phage display systems are generally based on N-terminal fusions to the coat proteins pIII or pVIII. pIII is present at 5 copies per virion, of which all 5 can be fused to short peptides without interfering with phage infectivity. The major coat protein pVIII is present at ~2700 copies per virion, of which ~10% can be reliably fused to peptides or proteins. As a result, peptides expressed as pIII fusions are present at low valency (1-5 copies per virion), while pVIII fusions are present at high valency (~200 copies per virion). The increased avidity effect of high valency pVIII display permits selection of very low affinity ligands, while low valency pIII display limits selection to higher affinity ligands. The Ph.D.™ Phage Display Cloning system is designed to make pIII fusions (5 copies of the peptide per virion). NEB’s pre-made libraries are the pentavalent pIII display format. Q: Does the Ph.D.™ Phage Display Cloning System come with double-stranded DNA vector? A: Yes, M13KE is supplied in RF, or double-stranded, form in #E8101S that may be used directly in restriction digest. Single-stranded M13 DNA may be obtained by PEG\/NaCl precipitation of phage particles from infected culture supernatants. Double-stranded DNA is purified from infected cells directly. A related product is the M13KE phage (#N0316S). Please see the Ph.D.™ Phage Display Manual for general M13 protocols. Q: What type of strain do I use with the Ph.D.™ Phage Display Cloning System? A: Any robust F’ or male E. coli strain may be used with M13. We prefer ER2738 (NEB #E4104S) or NEB Turbo (NEB #C2984). Cells may be made competent or purchased that way. For the most complex libraries, electrocompent cells must be used (eff. ≥109 transformants per ug M13). See our protocol for making electrocompetent cells in the Ph.D.™ Phage Display manual or purchase from NEB’s line of electrocompetent cells. Q: I have done all the standard troubleshooting for my cloning and transfection steps and I still cannot get the desired clone in M13KE? A: Make sure the pIII leader sequence is intact and in-frame with pIII coat protein in your design (see also cDNA FAQ#1). Assuming, the reading frame is correct, the insert may not allow the production of viable phage either due to issues with export into the cell periplasm or function of pIII coat protein after phage assembly. Inserts larger than 25-50 amino acids and those with N-termnal cysteines or positively charged residues are known to be problematic. These issues may be probed by subcloning the insert into another vector with KpnI\/EagI sites. For example, try to clone the insert into pMAL pIII (#N8101S) to see if the insert-MBP fusion can be produced and exported to the periplasm. Q: Where can I find the entire genome sequence for the parent vector, M13KE, used to clone Ph.D. Peptide Display Libraries? A: The DNA Sequences and Maps Tool page has the nucleotide sequence in several formats as well as a map of M13KE. Note, NEB #E8101S contains double-stranded, also known as replicative form (RF), M13KE DNA (20 ug). ","brand":"New England Biolabs","offers":[{"title":"Default Title","offer_id":46835523616937,"sku":"E8101S","price":0.99,"currency_code":"USD","in_stock":true}],"url":"https:\/\/iright.com\/products\/neb-e8101s","provider":"Iright","version":"1.0","type":"link"}