Restriction enzyme Hindlll

Prepare digests of DNA samples following the general format below 85 pL DNA solution, 10 pL 10X reaction buffer, 5 pL restriction enzyme (Hindlll) (10-20 U/pg DNA), for a total 100 pL of reaction volume. Tap tubes to mix and pulse centrifuge (5 s). Incubate digests for 2 h at 37°C. 

This procedure was empirically determined and may be optimized. The restriction enzyme Hindlll was chosen because the recognition sequence is absent from the transposon but is relatively frequent in B. burgdorferi DNA. 

Alkaliphilic Bacillus sp. AM-001 was aerobically grown to the early stationary phase at 37°C in alkaline medium (pH 9.0) containing 1% konjak mannan. Total chromosomal DNA obtained by the method of Saito and Miura( 14) was digested with Hindlll restriction enzyme. And 2 to 4 kbp DNA fraction of chromosomal DNA was collected by 1% agarose gel electrophoresis. The plasmid pUC19 was digested with Hindlll and then dephosphorylated with bacterial alkaline phosphatase. After the... 

Kits and enzymes Superscript reverse transcriptase (Invitrogen), Maxiscript and Megascript in vitro transcription kits (Ambion, Austin, TX). Taq-DNA polymerase, T4-polynucleotide kinase, EcoRI and Hindlll restriction enzymes (Invitrogen). 

Cut the constant regions using the restriction enzymes EcoRI and Hindlll. 

Extract the recombinant plasmids (pBSARs) (17) and double digest 5 pE (about 1 pg) of each of them with 1-5 U of EcoRI and Hindlll restriction enzymes (see Note 5). 

Restriction endonucleases are named in accordance with the proposals of Smith and Nathans (1973). Their names consist of a three letter abbreviation for the host organism (e.g. Eco for E. coli, Hin for Haemophilus influenzae. Bam for Bacillus amyloliquefaciens) followed by a strain designation and a Roman numeral. Thus Haemophilus influenzae, strain Rd contains at least three distinct restriction endonucleases and these are called Hindi, Hindll and Hindlll respectively. Restriction enzymes are generally known and described in the catalogues of suppliers by this code. 

The purity of the DNA can be assayed by assessing the susceptibility of the DNA to restriction enzymes. This can be done by running three forms of the isolated DNA on a minigel. The first is untreated DNA, the second is DNA treated with proper amounts of a common restriction enzyme (e.g., Hindlll or iscoRI), and the third sample is the DNA plus 0.25 to 0.5 fig of A DNA treated with a common restriction enzyme. If the DNA is pure enough for RFLP analysis or PCR, the cut DNA will show a smear compared to the uncut DNA. The A DNA plus isolated DNA sample should show a smear plus the A pattern overlaid on the smear. If the DNA is not pure, high molecular weight DNA will appear in all three lanes. 

BamRl, Hindlll, Afllll restriction enzymes, and buffers. 

Dpnl, Mbol, and Hindlll restriction enzymes and buffers. 

Figure 4. Construction of DNA fragments for atfl gene disruption and one step gene disruption. The restriction enzymes used were Hindlll (H), Clal (C), coRI (E), BamRl (B), Pst (P), Bg/II(Bg), Sail (S), Sphl (Sph). The position and direction of the open reading frame are indicated an arrow.

Figure 10. Map of the yeast ILV2 gene within a cloned DNA segment. Letters indicate restriction enzyme cleavage sites C, Clal V, EcoRV B, Bglll K, Kpnl R, EcoRI P, PvuII H, Hindlll. Deletions are indicated by bars, whereas lollipops indicate the sites of Tn 5 insertions. Mutations represented by filled symbols destroy ILV2 activity, whereas those depicted as open symbols do not affect ILV2 function. The extent of ILV2 mRNA is indicated by the wavy arrow.

Figure 1. An overlapping restriction map of the BOAA-degrading plasmid, pBYAl with respect to restriction enzymes Notl, Kpnl Hindlll, and Pstl.

Southern blot analysis of undigested (0) and restriction enzyme digested total Synechocystis DNA (1 Hindlll, 2 EcoRI,... 

Figure 1. Southern blot analysis of soybean genomic DNA. Probes used are. aCT cDNA (A) and BCCP cDNA (B). Restriction enzymes used for digestions are (1) -Nde1 (2) - Asel (3) - Bsu36 1 (4) EcoRV (5) - Avr II (6) - Afl II. All hybridizations were run at 60°C. Positions of lambda/Hindlll markers are shown. Presented data suggest multiple gene patterns for both subunits.

Fig. 2. Restriction enzyme map of the cloned 8.6-kb Hindlll fragment containing the gl8 locus and alignment of the sequence with the 0.8 kb gl8 cDNA. C=Clal, ti=Hindlll, P=Pstl, S=5fl/I, Sc=5ad, X=Xhol.

Low ionic strengths and high pH (8.0-9.5) alone or in combination with organic solvents and/or Mn generally favor the appearance of the star activity (see EcoRI, Section II,B)- The star activity has been documented in a number of restriction enzymes, including BamHl, BstI, Bsul, EcoRI, EcoRV, Hhal, Hindlll, PvuW, Seal, and Taql. Haelll, the isoschizomer of Bs RI, does not show any star activity under the same conditions for BsuKl. ... 

Restriction enzymes also display different sensitivities to noncanonically hemimethylated DNAs which are created, for example, by incorporating m C during the second-strand DNA synthesis catalyzed by DNA polymerases (40). At high enzyme-to-substrate ratios, some enzymes (e.g., Accl, BawiHl, Pstl, Sail, Smal, Sstl, and Xhol) are unable to cleave their recognition sites, whereas other enzymes (e.g., EcoRl, Hindlll, Dpnl, Pvull, and Xbal) are only partially sensitive to hemi-methylation. 

There is a systematic way in which restriction enzymes are named (Smith and Nathans, 1973). The first three letters are in italics, and represent the genus and species names of the host bacterium. A nonitalicized letter may appear next, representing the particular strain from which the isolate was purified. The name will end with a roman numeral to indicate the order of discovery for multiple restriction enzymes arising from the same bacterium. For example, coRI was isolated from Escherichia coli RY13, while Hindlll was isolated from Haemophilus influenzae strain d, and was the third enzyme isolated from this strain. (Note that the strain for Hindlll can be found in older pubUcations listed as Rd. The R is part of an R-M system, which denotes Restriction endonuclease versus Modification enzymes. The R and M strain designations are routinely dropped.)... 

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