Restriction enzyme

100% ethanol (1:0.75 v/v), TE buffer (10 mM Tris, 1 mM EDTA pH 8.0), 1.2% agarose gel (Agarose, 1X TAE buffer), 6X loading buffer (glycerol, Tris/EDTA pH 8.0, ethidium bromide), .25X TAE buffer, Restriction enzymes and Restriction endonuclease buffers. All the chemicals used were quality grade. The restriction

The first agarose gel provided insight into whether or not Zassy’s genomic DNA had been cut by the Sal I HF (NEB) restriction enzyme. As seen in Figure 1, Zassy’s cut DNA, in lane 3 left clear fragments between the 3 kb and 0.5 kb areas. Zassy’s uncut DNA, in lane 2, left a clear and bright band above the 10 kb marker. Therefore, the gel confirmed that Zassy’s DNA was successfully cut with Sal I because there was a significant difference the cut and uncut DNA that was run through the gel. There was

Fig5: Standard curve diagram of the concentration of BSA measured at an absorbance of 595 nm. The volume of each sample for restriction digest was calculated using the formula; C1V1 = C2V2 Where C1 = Initial Concentration V2 = Initial Volume C2 = Final Concentration V2 = Final Volume

(1). Restriction Digestion of Unknown Vector An unknown vector (0.1 µg/µL) was assigned to the group for restriction enzyme digestions. The vector could be either pUWL 500 or pUWL 501.Three enzymes, Pst I, Bgl II, and Eco RV, and their combinations were used to single, double and triple digest the unknown vector. A total of nine tubes of digestion mixtures were made for this experiment. Each digestion mixture contained 5 µL of unknown plasmid, 1.5 µL NEB Buffer, 1 µL of the restriction enzyme or

Gene Regulation for Safety Assessment: In addition to the mph gene, few gene regulations are implemented as safety assessment for the product. In the recombination process of making this product, Ecoli plasmid has been used as a vector to insert mph gene into host bacteria. We use a plasmid vector from E.coli that has conserved sequence for Plac promoter and gef suicide gene. Plac promoter is positioned upstream of mph gene to regulate its expression. Later, Plac promoter will be activated by induction

Restriction Analysis Hayley Keller Biochemistry Laboratory: BIOL 2324 TA: Manasa Madasu Date Performed: July 14th, 2015 Date Due: July 21st, 2015  Introduction: Restriction digest involves the use of restriction enzymes (also known as restriction endonucleases) to locate specific base pair sequences in DNA. These enzymes cut, or cleave, DNA only at their designated sequence, which is referred to as a recognition sequence. While there are four different types of restriction enzymes (1), the only

Restriction enzymes, also known as restriction endonucleases, are found naturally in some bacterial cells and are used in a restriction digest to produce smaller fragments of DNA by cutting the DNA. The enzymes work by cutting sequences on the DNA that are four to eight base pairs long. Restriction enzymes cut DNA on both strands and produce either blunt or sticky ends depending if the enzyme cuts right in the middle of the sequence (blunt end) or if the enzyme cuts and produces overhangs in the

Discussion In this experiment two methods of restriction mapping site were used i.e. double digestion method and the comparison of fragment pattern to a known sequence. In the double digestion method, the fragments produced by the enzyme Hind III andXhoI independently and concurrently were compared so that it can produce an estimation of relative postion of cleavage site The drawback of restriction mapping is that the technique cannot be directly used for eukaroyotes because of difficulties associated

Digestion of Restriction Enzymes BamHI and HindIII in Bacteriophage Lambda DNA and the Unknown Identification of the enzymes via Agarose Gel Electrophoresis BIOL3110L (February 2017) Elizabeth Phillips, University of Georgia, Athens, GA, USA ABSTRACT Agarose gel electrophoresis is used to identify the digestion of HindIII and BamHI restriction enzymes in bacteriophage lambda DNA. Restriction enzymes are the molecular scissors of biology and are used to separate DNA fragments. Unknown restriction enzymes

Background Amplified luxAB genes of isolated chromosomal DNA from Vibrio fischeri bacteria were ligated into digested pGEM [1]. The cut pGEM contained a “stuffer” – a small fragment of DNA positioned between the EcoR1 and Xba1 restriction sites and located within the polylinker region of the plasmid [1]. The stuffer fragment could potentially re-ligate back into the plasmid instead of the desired insert, namely the digested PCR product with luxAB genes [1]. Ligation reactions varied in two ways: