molecule at a particular place called restriction sites. Restriction enzymes also recognize a specific sequence of nucleotides‚ which vary between 4 and 8 nucleotides‚ in particular‚ palindromic. These restriction enzymes were kept in a 50% glycerol buffer that does not freeze at -20 oC. The four available enzymes used for this experiment were EcoRI‚ AvaI‚ HincII‚ and RsaI. The lengths of the DNA fragments from the restriction digests are used to map the relative locations of the four available enzymes
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Materials Restriction endonucleases Agarose TBE buffer Blue stain
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through. The shorter molecules migrate faster than the longer molecules. The use of electrophoresis buffer in the making of the agarose gel is to establish a constant pH and to provide ions to support the conductivity. There are three common buffers used in agarose gel electrophoresis. They are Tris-Acetate-EDTA (TAE)‚ Tris-Borate-EDTA (TBE) and Tris-phosphate. For this experiment‚ the best choice of buffer to use is TAE. The result of our experiment showed no bands. The reasons can be whether the concentration
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Introduction The purpose of this lab is to implement the technique of gel electrophoresis in the purification and size determination of various proteins and DNA fragments. In order to do this‚ a polyacrylamide gel will be prepared and placed in a buffer-containing gel electrophoresis apparatus. Next‚ an aliquot of acid phosphatase and a molecular weight marker (Composed of Phosphorylase B‚ bovine serum albumin‚ ovalbumin‚ and carbonic anhydrase) will be placed into separate wells within the gel‚
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Title: DNA analysis Aim: a) Isolate and Purify Bacterial Chromosomal DNA from a strain of E.coli b) Visualization of restriction fragments by Agarose Gel electrophoresis Objectives: * to isolate and purify bacterial chromosomal DNA from a strain of E.coli * to analyze and identify DNA by use of a spectro-photometer * to use restriction enzymes to cleave DNA into fragments * to visualize the restriction fragments by gel electrophoresis * to compare the different
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liquid nitrogen until the root tips turn to fine powder form. Next is the degradation of RNA step by adding 400 µl Buffer AP1 RNase to the sample in eppendorf tube. The mixture is incubated at 65°C for 10 minutes period after being vortex occasionally. In this period of time‚ the tube will be inverted 2-3 times during incubation. Then lysis step will take over by adding 130 µL Buffer P3. The solution then will be incubated for 5 minutes on ice until the tissue are completely lysed. The lysate solution
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will makes the plasmid which contains desired gene more easily to be isolated from self-ligated plasmid. The bacteria plasmid DNA that being extracted is from E. coli by applying miniprep method. Solution I‚ Solution II‚ Solution III‚ ethanol and TE buffer are miniprep reagent. The inoculums of E. coli was undergoes the first centrifugation under 12‚000rpm to separate out the
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PRECAUTION Methylated Spirit (ethanol‚ methanol‚ pyridine) Harmful vapour Harmful to skin Irritant to eyes Ventilate room Wear gloves Wear goggles Dyes (bromophenol blue‚ xylene cyanol‚ ponceau 4R‚ orange G) Irritant (xylene cyanol) Wear gloves TBE buffer (boric acid‚ EDTA‚ tris base) Harmful Wear gloves Agarose Irritant (eyes‚ powder) Handle carefully Wear gloves Wear goggles Detergent Not significant - Salt Not significant - Notes: none. Declaration - I have read and understood
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PCR or polymerase chain reaction was developed by Kary Mullis in 1983 and can amplify one or several strands or sections of DNA to millions or even billions in a matter of hours. PCR involves a DNA sample‚ primers‚ polymerases‚ DNA nucleotides‚ a buffer and a thermocycler. The primers are complementary region to certain stretches of DNA. They mark the boarders of the DNA region one wishes to amplify and provide a place for a polymerase to bind. The standard DNA polymerase now used in PCR is isolated
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cycler or... For manual cycling • Water baths‚ 3 (maintained at 94‚ 55 and 72 °C) • Floating holder for microcentrifuge tubes • Stopclock For the electrophoresis • Agarose solution‚ (1.5% in TBE buffer‚ melted in a microwave oven then kept molten in a water bath or incubator at 55–60 °C) • TBE buffer • Azure A solution for staining the gel (0.04% in 20% ethanol) • Micropipette or similar device‚ with a 5–40 μl range www.bioscience-explained.org 1 COPYRIGHT © BIOSCIENCE EXPLAINED‚ 2002 bioscience
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