pcr grade nucleotide mix  (Thermo Fisher)

Journal: PLoS ONE

Article Title: Mechanism of Human Papillomavirus Binding to Human Spermatozoa and Fertilizing Ability of Infected Spermatozoa

doi: 10.1371/journal.pone.0015036

Figure Lengend Snippet: a . Scheme of the recombinant plasmid pIRES2-AcGFP1-E6E7. E6/E7 genes have been amplified (1032 bp) from plasmid p1321 HPV-16 E6/E7 by PCR and subcloned to plasmid pIRES2-AcGFP1 between SalI and BamHI restriction sites. b . PCR for HPV E6/E7 genes from transfected sperm. Lane M: DNA marker (100 bp); 1: sperm transfected with recombinant E6/E7 plasmid; 2: negative control (no template); 3: sperm transfected only with Lipofectamine 2000; 4: positive control (pIRES2-AcGFP1-E6E7 plasmid). c . Mean number of human sperm penetrated per hamster oocyte in control and sperm transfected with HPV-16 E6/E7 plasmid. d . Hamster oocytes penetrated by control sperm and sperm transfected with HPV16 E6/E7 plasmid in bright field (BF, upper panel) and fluorescence (FL, lower panel) using SYBR green DNA stain.

Article Snippet: The PCR mixture consisted of 5 µL Expand high fidelity buffer with MgCl 2 , 1 µL of PCR grade nucleotide mix (10×), 20 pmol of each primer with SalI and BamHI restriction sites (New England Biolabs, Ipswich, MA) including forward: 5′-GTGCGCGTCGACGTGATGCACCAAAAGAGAACTG-3′ and reverse: 5′-GTGCGCGGATCCGTGTGGTTTCTGAGAACAGATG-3′ , 0,75 µL Expand high fidelity enzyme mix (Expand High Fidelity PCR System dNTPack, Roche Applied Science, Mannheim, Germany) and sterile H 2 O in a final volume of 50 μL.

Techniques: Recombinant, Plasmid Preparation, Amplification, Transfection, Marker, Negative Control, Positive Control, Fluorescence, SYBR Green Assay, Staining