KpnI restriction enzyme

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	In the current study, the ACAT1 gene P1 promoter was isolated from the pMD19-T-P1 vector following digestion with KpnI/XhoI, and the fragment was subcloned into the multiple cloning sites of the Firefly luciferase reporter gene vector pGL3-Enhancer to obtain P1E-1.

Protocol tips
In the current study, the ACAT1 gene P1 promoter was isolated from the pMD19-T-P1 vector following digestion with KpnI/XhoI, and the fragment was subcloned into the multiple cloning sites of the Firefly luciferase reporter gene vector pGL3-Enhancer to obtain P1E-1.

Publication protocol

The previously described cloning vector pMD19-T-P1 constructed in our laboratory (9) containing the −603/+65 region of human ACAT1 gene P1 promoter was used in this study. In the current study, the ACAT1 gene P1 promoter was isolated from the pMD19-T-P1 vector following digestion with KpnI/XhoI, and the fragment was subcloned into the multiple cloning sites of the Firefly luciferase reporter gene vector pGL3-Enhancer to obtain P1E-1.Transcription Element Search System (www.cbil.upenn.edu/cgi-bin/tess/tess) was used to predict transcription factor binding sites in the DNA sequence of the human ACAT1 P1 promoter. According to the analysis of biological information, the P1E-1 plasmid was used to generate deletions of the ACAT1 gene P1 promoter with varying 5′ ends and an identical 3′ end at +65 by polymerase chain reaction (PCR). The primer sequences, presented in Table I, were designed according to the human ACAT1 gene P1 promoter GenBank sequence (www.ncbi.nlm.nih.gov/genbank; GenBank accession no. AF143319), all included a 6-bp KpnI/XhoI linker. All PCR reactions were performed in a total volume of 50 µl containing 1X Pyrobest buffer II, 1.5 mmol/l MgCl2, 200 µmol/l deoxynucleotides, 200 nmol/l each primer, 2 units Pyrobest DNA polymerase and 0.5 µg DNA. Cycling conditions were as follows: 23 Cycles of pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 sec, annealing at 52°C for 30 sec, and extension at 72°C for 1 min (Bio-Rad T100 PCR Thermal Cycler; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The PCR products were then digested with KpnI and XhoI. The digested PCR products were analysed using agarose gel electrophoresis and then retrieved using a gel extraction kit (Axygen Biosciences). The pGL3 Enhancer plasmid was also digested by double digestion and the large fragment was retrieved. During a ligation reaction, the recycled PCR products and empty pGL3 Enhancer vector were incubated overnight with T4 DNA ligase at 4°C, transformed into E. coli DH5α (Takara Biotechnology Co., Ltd.) and screened. The plasmid was extracted and identified by PCR amplification, restriction enzyme digestion mapping and DNA sequencing. The 5′-deletion constructs of the P1 promoter represent bases −547 to +65 bp (P1E-2), −498 to +65 bp (P1E-3), −428 to +65 bp (P1E-4), −363 to +65 bp (P1E-5), −324 to +65 bp (P1E-6), −256 to +65 bp (P1E-7), −188 to +65 bp (P1E-8) and −125 to +65 bp (P1E-9). These constructs were then identified by restriction enzyme digestion mapping and DNA sequencing. Sequences were determined on an ABI 3730 automated DNA sequence analyzer by Invitrogen (Thermo Fisher Scientific, Inc.). The plasmids were purified by the Wizard purification plasmid DNA purification system (Promega Corporation, Madison, WI, USA) to transfect cells.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Restriction Enzymes KpnI using KpnI restriction enzyme from Takara Bio Inc.

Manufacturer protocol

Download the product protocol from Takara Bio Inc for KpnI restriction enzyme below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Restriction Enzymes KpnI using KpnI restriction enzyme from Takara Bio Inc. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.