Pgem t easy cloning vector

SCAL1 gene blocks were obtained from Integrated DNA Technologies (Coralville, IA, USA) (ref.no. 98409609). This transcript was cloned into the pGEM^®-T Easy cloning vector (Promega^®) via non-directional TA-cloning. A-overhangs were added prior to ligation as follows: a tube containing 1× Titanium Taq buffer, 5× Titanium Taq polymerase (Clontech Laboratories, Inc., Mountain View, CA, USA), and 1 mM dNTPs was first incubated at 95 °C for 5 min, after which 24.5 ng of the gene block was added and the resulting mixture was incubated at 72 °C for 15 min. From the solution with poly A-appended SCAL1 blocks, 1.1 µl was added to a separate mixture of 2× T4 DNA ligase buffer (Roche^® Diagnostic, GmbH, Mannheim, Germany), 50 ng of pGEM^®-T Easy cloning vector (Promega^®), and 0.25 U of T4 DNA ligase buffer (Roche^®). The 5.2-µl solution was then incubated with the following profile: 4 °C for 2 h, 22 °C for 4 h, and 14 °C overnight after which it was used for transformation into DH5α ultracompetent E. coli cells. Recombinant pGEM^®-T Easy-SCAL1 constructs with verified sequences were digested with EcoRI, subcloned into the pTargeT™ mammalian expression vector, and verified by sequencing for accuracy and directionality.

Amplified product of ovine CD14 was checked by 1% agarose gel electrophoresis. The products were purified from gel using Gel extraction kit (Qiagen GmbH, Hilden, Germany).pGEM-T easy cloning vector (Promega, Madison, WI, USA) was used for cloning. Then, 10μL of ligated product was mixed thoroughly to 200μL competent cells, and heat shock was given at 42°C for 45sec in a water bath. Subsequently, the cells were immediately transferred on chilled ice for 5 min., and SOC medium was added to it. The bacterial culture was centrifuged to obtain the pellet and plated on an LB agar plate containing Ampicillin (100mg/mL) added to the agar plate @1:1000, IPTG (200mg/mL) and X-Gal (20mg/mL) for blue-white screening. Plasmid isolation from overnight-grown culture was carried out by small-scale alkaline lysis method as described in (54 (link)). Recombinant plasmids were characterized by PCR using CD14 primers as reported earlier and restriction enzyme digestion. CD14 gene fragments released by enzyme EcoRI (MBI Fermentas, USA) were inserted in recombinant plasmid which was sequenced by dideoxy chain termination method with T7 and SP6 primers in an automated sequencer (ABI prism, Chromous Biotech, Bangalore).

We cloned and sequenced full-length complementary DNA (cDNA) and partial genomic DNA (gDNA) of PgCad1 from fourth instar larvae of JL46 strain survivors on a diagnostic concentration of Cry1Ac (e.g. 10 μg Cry1Ac protoxin per mL diet). Total RNA and gDNA were extracted from fourth instar individual larvae (n = 8) using TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa). M-MLV Reverse Transcriptase (Promega) was used for first strand cDNA synthesis and full-length PgCad1 cDNA was PCR amplified using primers F1 + R1 and F2 + R2 (Table S1) Primers were designed based on full-length cDNA sequence of BtR-s allele (Genbank Accession Number AY198374.1) from APHIS-S. For cloning gDNA flanking the r15 mutation site, we used gF46 + gR46 (Table S1) and LA-Taq (TAKARA, Dalian, China) to PCR amplify the PgCad1 partial gDNA fragment. PCR conditions were: 94 °C for 2 min, followed by 32 cycles at 98 °C for 10 s, 60 °C for 30 s, and 72 °C for 3 min, and a final extension at 72 °C for 7 min. PCR products were cloned into pGEM®-T Easy Cloning Vector (Promega) cloning vector and DNA sequencing was performed as previously described^{35 (link)}.