NdeI in forward primer of YML1 and YML2, KpnI in forward primer of YML3 and YML4, BamHI
in reverse primer of YML 1 to 4, XhoI in YML5 forward primer, XbaI in YML5 reverse primer).
DNA in the plugs was digested with 40 U of XhoI (New England Biolabs, Ipswich, MA, USA) or 40 U of BamHI
(New England Biolabs), for 16 and 3 h, respectively, at 37[degrees]C following the manufacturer's protocol.
These PCR products were cloned into the EcoRI and BamHI
sites of the pLVX-Tight-Puro vector.
Digestion with BamHI
showed two patterns, RPOIILS-BamHI
pattern A with 98 and 150 bp fragments and RPOIILS-BamHI
pattern B without digestion.
Staying on at Khan Market is worth it, Bamhi
says, even if it means abandoning the comforts of his own car sometimes, because he doesn't want to take it out of its parking spot outside his shop.
The DNA encoding the N-terminal and C-terminal fragments of gp96 consisted of 1014 (from 64-1077 nucleotide) and 907 (from1645-2552 nucleotide) base pairs, respectively, were cloned into the BamHI
and KpnI sites of expression vector pQE30.
PCR-RFLP: Equal quantity of PCR products were digested for 3h with BamHI
, BstNI or HgaI (New England Biolabs, USA) at 37[degrees]C for BamHI
and HgaI and 60[degrees]C for BstNI following manufactures instruction.
The a subunit was amplified with PCR primers containing an XbaI site (GAT CTC TAG AAT GGC TCC TGC CAT GGA ATC) and a BamHI
site (GAT CGG ATC CTC AGT TGT TTT TGG GGT TTG GC), the [beta] subunit using primers containing a Not I site (GAT CGC GGC CGC TAT GGA CAC AGA AAG TAA TAG GAG) and an SfiI site (GAT CGG CCC AGC CGG CCT CAT AAA TCA ATG GGA GGA GAC ATT TC) and the [gamma] subunit using primers containing an NheI site (GAT CGC TAG CAT GAT TCC AGC AGT GGT CTT G) and a BamHI
site (GAT CGG ATC CCT ACT GTG GTG GTT TCT CAT GC).
Rosen-Wolff A, Ben-Hur T, Becker Y, Darai, G (1988) Comparative analysis of the transcripts mapped in the BamHI
DNA fragment B of avirulent HSV-1 HFEM, virulent HSV-1 F, and their intratypic recombinant viruses.
Then, the oligonucleotides 5'-GATCTATCGATTCTAGAGGATCCTCGAGATATCCC-3' and 5'-GGGATATCTCGAGGATCCTCTAGAATCGATG A-3' (containing BglII, ClaI, XbaI, BamHI
, XhoI, EcoRV) were ligated to this smaI-smaI fragment from pIND/Hygro and then digested with BglII and HindIII (about 300 bp, contains MCS and hs).
Ten micrograms of high-molecular-weight DNA was digested with 200 units of EcoRI, HindIII, and BamHI
(Promega, Madison, Wis) in the presence of 4 mmol/L spermidine (Sigma) and 100 mg/mL bovine serum albumin (Sigma) for 3 to 4 hours, precipitated in ethanol, and reconstituted in TE buffer (10 mmol/L Tris-HCl, pH 7.
A part of neo gene (sequence 2423 to 5111) was amplified from the pMAMneo vector (CLONTECH) using primers (5'-cgtaggatccgat ccggctg-3') and (5'-cgatgatatcccagacatga-3') and ligated to the BamHI
and EcoRV of the pBC10 vector.