Hindlll

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... 

Partial random DNA template (108 bp) (synthesized by Biosource International, Foster City, CA). 5 -ACC-CAG-TCC-ACA-AGC-TTG-TAG-TAC-TNN-NNN-NNN-NNN-NNN-NNN-NNN-NNN-NNN-NNN-NNN-NNN-NNN-NNC-CCT-AGA-TGG-CAG-TTG-AAT-TCT-CCC-TAT-AGT-GAG-TCG-TAT-TAC-3 (N=A, C, G, or T are incorporated with equal probabilities). Restriction sites (Hindlll and EcoRI) are underlined. 

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. 

Cut pGEM-3Z vector (Promega) using EcoRI and Hindlll and gel-purify the linearized vector. 

Transform into E. coli cells, prepare minipreps and identify correct clones by digesting with Sail and Hindlll. Correctly ligated clones show two bands of 1.6 and 3.0 kb, plasmids without insert show only the 4.3 kb vector band. M13 forward, Ml3 reverse, and bpA-for primers can be used for sequence confirmation. Prepare Maxipreps and use two independent plasmids of pRMCE-U6-shRNA for stable transfection by RMCE in ES cells. 

Hindered phenohcs Hindered phenols Hindll Hindlll Hinfl Hinosan Hipercos Hi-pHase... 

Calculate the frequency of occurrence of restriction sites for PstI and Hindlll in the DNA from a ther-mophile (80% G + C) and from E. coli (52% G + C). 

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