What bacteria were used for the isolation of the three enzymes (HindIII, NdeI and PvuI)? What type of ends do these restriction enzymes produce?notes are below Figure 4-1: Several examples of DNA molecular weight (MW) ladders used during agarose gelelectrophoresis. A. 2 log DNA ladder (Biolab). B. 1 kb ladder (NEB). C. 1 kb ladder (Invitrogen). D. 100 bpladder. E. A Hinduu digest.А.В.C.D.Е.Me RMas ing216tayta Mng491223,1300 20-205415--20144 12--1636247 erSlt106,517iar i antf the ecter2007Figure 4-2: PUC19 map restriction sites with a variety of enzymesDedl 91hen M 51Bem 2613BstAPI 179Ndel 183Kasl - Narl - Siol 235Eco010 2074Aatll - Zral 2817Bgll 245Fel 256Bkill 2542Pul 276Prull 308Benl 364Sepl 2501Al 22BeN 387Apel- Ee 306aall- Sacl - EoekKI 42Aerts1 - pal 40Xmal 2204laczakd 2215Scal 2177MCSwal- oN- Smal-Tip1- Naal 40n 47thalAecl - Hixl- Sal e0. al ePrul 2068wall 2059pUC 192,686 bprl 105Aell 1924he asinin 47Fl 1919Prull 628Aall 187All 1822Bgll 1813Bpnd 1714T 641Bull 659Bspl - Sapl 683Thil 781Bsrfl 1779hal 1765ell 1753AI - Peil 06Dal 208oriBnrl 1744Alll 1694kiM 1015RueYI 1110AlwNI 1217RceN 1221.書dy IntroductionThe analysis of DNA frequently requires determining the size of individual DNA molecules. Under mostconditions, DNA has a relatively uniform acidic or negative charge caused by the phosphate groups on itsouter surface. When placed in an electric field during electrophoresis, negatively charged molecules suchas DNA migrate toward the positive electrode. In a semisolid matrix like an agarose gel, larger DNAmolecules are retarded by the sieving properties of the matrix, so they move more slowly and migrate ashorter distance jo a giyen time than smaller DNA molecules. The migration rate of DNA molecules isinversely proportional to their molecular weights. (Agarose is a purified form of agar, a complexpolysaccharide derived from seaweed.)You will be performing restriction enzyme digests of your plasmid DNA samples and analyzing them on anagarose gel. Restriction endonudleases are enzymes that recognize, bind to and deave DNA atspecific nudeotide sequences. Approximately 300 different restriction enzymes are currently commerciallyavailable. Restriction enzymes occur naturally in all bacteria and are used to protect the bacteria frominvading foreign DNA (e.g. a virus) by digesting the foreign DNA at a specific recognition sequence. Mostenzymes recognize a 6-base pair (bp) sequence. The specificity of restriction enzymes makes them veryuseful tools in manipulating and cloning DNA molecules.In this experiment, you will be digesting your samples with the enzymes Ndel HindII, and Pvul.The recognition site for Ndel is 5'-CA/TATA G-3', the site for Hind is 5'-A/AGCTT-3' and the site forPuul is 5-CGAT/CG-3'. The enzymatic activity of restriction enzymes is measured in units of theiractivity. This activity varies between enzymes depending on their purification, source, and concentrationof the protein. The activity of a restriction enzyme is defined as its ability to cut DNA. Therefore, thedefinition of one unit (U) of restriction enzyme activity is:"The amount of restriction enzyme needed to completely digest (cut) one microgram (ug) of substrateDNA in one hour at the optimum temperature of the enzyme (usually 37 °C) in a 50 pl reaction volume."This definition is important and useful in calculating the amount of enzyme required to successfullycomplete a given restriction digest. One must use the appropriate number of units of a specific enzyme in.order.to completely digest the sample. It is also important to note that an excess of enzyme may result innon-specific or incomplete digestion of DNA. This is partly due to the glycerol used to stabilize theenzyme; therefore, the total amount of all enzymes used in the digest should never be more than 10%the total volume of the digest (e.n. 2 pl in 20 µL). On the other hand, impurities in the DNA sample mayinhibit digestion. Usually up to a 10-fold excess of each restriction enzyme is used to digest plasmid mini-prep DNA.DNA size standards must be run on every gel that you wish to measure DNA fragment size. The sizestandards can be marker DNA composed of a one kilobase "ladder", a 100-bp ladder or a lambda (A) DNAdigest (HinduL EcaRI or HindiItEcoRD(See photographs below; NEB.) The complete sequence ofmarker DNA s is known, and the sizes of DNA fragments have been precisely determined. This DNA iscommercially available and is relatively inexpensive. The bands of DNA are visualized by staining eitherwith the dye ethidium bromide* or the dye SybrSafe" which intercalate with DNA. When placed undera UV light source, the DNA samples will fluoresce. This signal can be detected by film and/or suitableimaging devices.

HindIII is isolated from Haemophilus influenzae . NdeI is an isolated from Neisseria…

Answered: What bacteria were used for the…

11th Edition

ISBN:9780134580999

Author:Elaine N. Marieb, Katja N. Hoehn

Publisher:Elaine N. Marieb, Katja N. Hoehn

Chapter1: The Human Body: An Orientation

Section: Chapter Questions

Problem 1RQ: The correct sequence of levels forming the structural hierarchy is A. (a) organ, organ system,...