Gentra Puregene Cell Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Frozen samples should be thawed and equilibrated to room temperature (15–25°C) before beginning the procedure.

Upstream tips
Frozen samples should be thawed and equilibrated to room temperature (15–25°C) before beginning the procedure.

Publication protocol

Exemplary electrophoretic mobility agarose gel shift assay
The promoter fragments (100–200 bp) harboring central MatInspector predicted (bipartite) DR3-type VDREs for CRABP2 (forward: 5′-CAAGCCCCTCTCTGAGTACG-3′, reverse: 5′-GATGCTCAGGGCTCGTGTAT-3′), OLFML3 (5′-TCCCAGTGGTAAGGAAGTCAG-3′, 5′-TTGCTTGAAAGAGGCCAGAT-3′), BNC2 (5′-GTCAAGCATGCCTGTGAAGA-3′, 5′-GGCCCTCCTTATTTCAGTCC-3′), TMEM91 (5′-CCACGATCACTTCACTGCAC-3′, 5′-GCCTTAACCAAGGACCTTCC-3′), and KRT19 (5′-CTGCTGTGAGGATCCAGTGA-3′, 5′-GTCTGGGGAGGGACTTTGTA-3′) were PCR amplified from genomic DNA isolated from cultured human keratinocytes (Gentra Puregene, Qiagen GmbH) with the Primer3-designed oligonucleotides listed in brackets. The VDR DNA-binding domain (DBD, AA 16–125) (Sone et al. 1991) was shuttled into recombinational cloning compatible destination vector pDEST 17 (Life Technologies), transformed into Escherichia coli BL21 (DE3) cells, expressed via 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) induction, and then purified as recombinant hexahistidine-tagged fusion protein by nickel affinity chromatography following common procedures. To investigate binding of VDR-DBD to amplified promoters, 600 ng of respective PCR products were incubated with recombinant DBD in 100 mM Tris buffer in a total reaction volume of 20 μl for 5 min and subsequently separated on a 1% agarose gel in 1× TAE buffer. Samples were visualized for apparent electrophoretic mobility shifts in relation to a previously described osteopontin DR3 repeat (Hijiya et al. 1994) positive control (5′-CGCAGAGCATTTGCATCTAA-3′, 5′-GGTTCTGAATTCCGCTGTGT-3′) plus four analogously treated negative control DNA fragments (designated as C7, D1, A12 and G8) containing no definite VDRE. Band intensities were densitometrically quantified via ImageJ software (http://rsbweb.nih.gov/ij/docs/index.html).

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 DNA isolation / purification Cells - Primary cells Human primary keratinocytes using Gentra Puregene Cell Kit from Qiagen.

Manufacturer protocol

Download the product protocol from Qiagen for Gentra Puregene Cell Kit below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing DNA isolation / purification Cells - Primary cells Human primary keratinocytes using Gentra Puregene Cell Kit from Qiagen. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.