S1 nuclease mapping of the 5’ end of a transcript.

Description:Calf Intestinal Alkaline Phosphatase (CIAP) is a phosphomonoesterase that removes 3´ and 5´ phosphates from DNA and RNA.

Applications: Dephosphorylation of 5´-phosphorylated termini of vector DNA to prevent self-ligation (1). Dephosphorylation of 5´ termini of nucleic acids prior to forward reaction with kinase.

Source: Purified from calf intestinal mucosa.

Performance and Quality Testing: Endodeoxyribonuclease, 3´ exodeoxyribonuclease, and ribonuclease assays; dephosphorylation efficiency measured in a transformation assay.

Unit Definition: One unit hydrolyzes 1 µmol of 4-nitrophenyl phosphate in 1 min. at 37°C.

Unit Reaction Conditions: 1 M diethanolamine buffer, 10 mM 4-nitrophenyl phosphate, 0.25 mM MgCl2 (pH 9.8) in 900 µl for 10 min. at 37°C.

Contents and Storage:Calf Intestinal Alkaline Phosphatase is supplied with a vial of 10X dephosphorylation buffer [500 mM Tris-HCl (pH 8.5), 1 mM EDTA], vial of dilution buffer [50% glycerol, 25 mM Tris-HCl (pH 7.6), 1 mM MgCl2 , and 0.1 mM ZnCl2 ]. Store Calf Intestinal Alkaline Phosphatase at -20°C.

Reference(s):1. Schmidt, B. et al. (1998) Focus 20: 52.

Source:www.invitrogen.com

Description: Bacterial Alkaline Phosphatase (BAP) removes 3´ and 5´ phosphates from DNA and RNA. BAP is active at 65°C for at least 1 h and is inactivated by phenol extraction.

Applications: Dephosphorylation of 5´-phosphorylated termini of vector DNA to prevent self-ligation. Dephosphorylation of 5´ termini of nucleic acids prior to forward reaction with kinase.

Source: Purified from E. coli C90.

Performance and Quality Testing: Endodeoxyribonuclease, exodeoxyribonuclease, and ribonuclease assays; dephosphorylationefficiency determined.

Unit Definition: One unit hydrolyzes 1 nmol of ATP in 30 min. at 37°C.Unit Reaction Conditions: 10 mM Tris-HCl (pH 8.0), 1.5 mM [-32P]ATP, and enzyme in 50 µl for 30 min. at 37°C.Contents and Storage:Bacterial Alkaline Phosphatase is supplied with a vial of 10X dephosphorylation buffer [100 mM Tris-HCl (pH 8.0)]. Store at -20°C.

Source:www.invitrogen.com

Description:T4 Polynucleotide Kinase catalyzes the transfer of the -phosphate of ATP to the 5´ hydroxyl terminus of DNA or RNA.A T4 Polynucleotide Kinase Technical Bulletin is available.

Applications: 5´ end-labeling of DNA or RNA (1). Phosphorylation of synthetic linkers (1).

Source: Purified from E. coli expressing the T4 Polynucleotide Kinase gene in a vector.

Performance and Quality Testing: Ribonuclease, endodeoxyribonuclease, 3´ and 5´ exodeoxyribonuclease, and phosphatase assays;labeling efficiency tested.

Unit Definition: One unit transfers 1 nmol of phosphate of ATP to the 5´ hydroxyl termini of micrococcal nuclease-treated DNA in30 min. at 37°C.

Unit Reaction Conditions: 70 mM Tris-HCl (pH 7.6), 0.1 M KCl, 10 mM MgCl2 , 1 mM 2-mercaptoethanol, 0.3 mM [-32P]ATP,0.38 mg/ml micrococcal nuclease-treated DNA, and enzyme in 150 µl for 30 min. at 37°C.

Contents and Storage:T4 Polynucleotide Kinase is supplied with a vial of 5X forward reaction buffer [350 mM Tris-HCl (pH 7.6), 50 mM MgCl2 , 500 mM KCl, 5 mM 2-mercaptoethanol] and a vial of 5X exchange reaction buffer [250 mM imidazole-HCl (pH 6.4), 60 mM MgCl2 , 5 mM 2-mercaptoethanol, 350 µM ADP]. Store at -20°C.

Reference(s):1. T4 Polynucleotide Kinase Technical Bulletin.

Description:S1 Nuclease is a single-strand-specific endonuclease that hydrolyzes single-stranded RNA or DNA into 5´ mononucleotides. The enzyme will hydrolyze single-stranded regions in duplex DNA such as loops and gaps. S1 Nuclease is stable at 65°C.

Applications: Nuclease mapping techniques (1,2). Removal of single-stranded regions from double-stranded DNA (3). Exo III-ordered sequencing (4).Source: Isolated from Aspergillus oryzae.

Performance and Quality Testing: Double-strand-specific deoxyribonuclease and phosphatase assays.

Unit Definition: One unit hydrolyzes 1 µg of denatured DNA to acid-soluble material in 1 min. at 37°C.

Unit Reaction Conditions: 30 mM sodium acetate (pH 4.6), 50 mM NaCl, 1 mM zinc acetate, 0.5 mg/ml heat-denatured DNA, 5% (v/v) glycerol, and enzyme in 0.5 ml for 10 min. at 37°C.

Contents and Storage:S1 Nuclease is supplied with a vial of 10X S1 Nuclease buffer [300 mM sodium acetate (pH 4.6), 10 mM zinc acetate, 50% (v/v) glycerol], vial of dilution buffer, vial of 3 M NaCl. Store at -20°C.

Reference(s):1. Berk, A.J. et al. (1977) Cell 12: 721.2. Berk, A.J. (1981) Focus® 3: 1.3. Gerard, G.F. (1985) Focus® 7: 7.4. Hoheisel, J. et al. (1986) Nucleic Acids Res. 14: 3605.

S1 mapping of the 3’ end of a transcript

Sanger sequencing of

DNA

Figure 5.19Sanger

sequencing of DNA

Sanger sequencing of DNA

Figure 5.20b

Sequence read

Primer extension

Description:SuperScript™ Reverse Transcriptase (RT) is a DNA polymerase that synthesizes a complementary DNA strand from single-stranded RNA, DNA, or an RNA:DNA hybrid. This enzyme is produced from a cloned M-MLV RT gene from which the RNase H sequence has been deleted, reducing the RNase H activity. This structural modification prevents degradation of the RNA molecule during first-strand cDNA synthesis (Figure 1).

Purified from E. coli expressing the pol gene of M-MLV (4,5), mutagenized to reduce the RNase H activity (6).

Unit Description:One unit incorporates 1 nmol of deoxyribonucleotide into acid-precipitable material in 10 min. at 37°C using poly(A)•oligo(dT)12–18 as template•primer (7).

Unit Reaction Condition:50 mM Tris-HCl (pH 8.3), 40 mM KCl, 6 mM MgCl2, 1 mM DTT, 0.5 mM [3H]dTTP, 0.1 mM poly(A), 0.1 mM oligo(dT)12–18, 0.1 mg/ml BSA, and enzyme in 50 µl for 10 min. at 37°C.

Contents and Storage:SuperScript™ RNase H- Reverse Transcriptase is supplied with a vial of 5X first-strand buffer [250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2], vial of 100 mM DTT. Store at -20°C.

Reference(s):1. Gerard, G. (1989) Focus® 11: 66.2. Howland, P. et al. (1991) Mol. Brain Res. 11: 345.3. Gerard, G.F. et al. (1993) in Methods in Molecular Biology, Vol 16: Enzymes of Molecular Biology (Burrell, M. Ed.), Humana Press, Totowa, N.J., p. 73.4. Kotewicz, M. et al. (1985) Gene 35: 249.5. Kotewicz, M.L. et al. (1988) Nucleic Acids Res. 16: 265.6. Houts, G. et al. (1979) J. Virol. 29: 519.

Run-off transcription

Nuclear run-on transcription

The cat reporter gene to measure transcription

Accumulation of acetylated CAM

Nitrocellulose filter binding assay

Gel mobility shift assay

DNase footprinting

Next generation DNA sequencing

Solexa sequencing

Solexa sequencing

Solexa sequencing

Solexa sequencing

454 sequencing

454 sequencing