RECOMMENDATIONS FOR USE ADDITIONAL PROTOCOLS

Taq DNA Polymerase or Component DreamTaq DNA Polymerase 10X Taq buffer dNTP Mix, 2mM each 25 mM MgCl2 M13/pUC Reverse Sequencing primer (#SO101) M13/pUC Forward Sequencing primer (#SO100) Taq DNA polymerase PCR Master Mix (2X) Water, nuclease-free Total volume 0.1 l (0.5 u) 0.6 l (10 M) 0.6 l (10 M) 0.6 l (10 M) 0.6 l (10 M) 2 l 2 l 1.2 l PCR Master Mix (2X)

10 l

to 20 l 20 l

to 20 l 20 l

Analysis of Recombinant Clones

Analyze 4-6 white colonies for the presence and orientation of the DNA insert using one of the following methods. Restriction analysis. Isolate plasmid DNA from an overnight bacterial culture using a convenient plasmid miniprep method such as GeneJET Plasmid Miniprep Kit. Use FastDigest restriction enzymes to digest DNA from recombinant clones in just 5 min. Sequencing. Isolate plasmid DNA from an overnight bacterial culture using a reliable plasmid miniprep method such as GeneJET Plasmid Miniprep Kit. Sequence the insert using appropriate sequencing primers. Colony PCR. Use the following protocol for colony screening by PCR. 1. Prepare enough PCR master mix for the number of colonies analyzed plus one extra. For each 20 l of reaction, mix the following reagents: 2. Mix thoroughly, spin briefly and aliquot 20 l of the mix into the PCR tubes on ice. 3. Pick up an individual colony with a sterile pipette tip and resuspend it in 20 l of the PCR master mix. Make a short strike with the same tip over culture plate to save the clone for repropagation. 4. Perform PCR: 95C, 3 min; 94C, 30 s, 45C*, 30 s, 72C 1 min/kb; 30 cycles. 5. Analyze on a gel for the presence of the PCR product of the expected length. * Depends on primer pair used (Tm-5). In vivo DNA Transfection using TurboFect in vivo Transfection Reagent

DNA ligation with T4 DNA Ligase (M0202)

Protocol

1. Set up the following reaction in a microcentrifuge tube on ice. (T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert.) COMPONENT 20 l REACTION

10X T4 DNA Ligase Buffer* 2 l Vector DNA (3 kb) Insert DNA (1 kb) Nuclease-free water T4 DNA Ligase 50 ng (0.025 pmol) 50 ng (0.076 pmol) to 20 l 1 l

2. * The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature. 3. Gently mix the reaction by pipetting up and down and microfuge briefly. 4. For cohesive (sticky) ends, incubate at 16C overnight or room temperature for 10 minutes. 5. For blunt ends or single base overhangs, incubate at 16C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation). 6. Chill on ice and transform 1-5 l of the reaction into 50 l competent cells.

Description: New England Biolabs provides a color-coded 10X NEBuffer with each restriction endonuclease to ensure optimal (100%) activity. Most of our enzymes are supplied with one of four standard NEBuffers. Occasionally, an enzyme has specific buffer requirements not met by one of the four standard NEBuffers, in which case the enzyme is supplied with its own unique NEBuffer. Some restriction endonucleases require BSA at a final concentration of 100 g/ml for optimal activity. When required, BSA is supplied as a 10 mg/ml (100X) stock and should be added to the reaction mixture. Reagents Supplied: 10X NEBuffer 1 BSA (100X) 1X NEBuffer 1: 10 mM Bis-Tris-Propane-HCl 10 mM MgCl2 1 mM Dithiothreitol pH 7.0 @ 25C

Activity in NEBuffers: NEBuffer 1: 75% NEBuffer 2: 100% NEBuffer 3: 100% NEBuffer 4: 100%

When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion.

Methylation Sensitivity:

dam methylation: Not sensitive dcm methylation: Not sensitive CpG methylation: Impaired More information about: Methylation Sensitivity Heat Inactivation: 65C for 20 minutes

Survival in a Reaction: (+ + +) Suitable for an extended or overnight digestion. Enzyme is active > 8 hours. More information about: Extended Digests with Restriction Enzymes

Reaction & Storage Conditions

Reaction Conditions: 1X NEBuffer 4 Supplemented with 100 g/ml Bovine Serum Albumin Incubate at 37C. 1X NEBuffer 4: 20 mM Tris-acetate 50 mM potassium acetate 10 mM Magnesium Acetate 1 mM Dithiothreitol pH 7.9 @ 25C Unit Definition: One unit is defined as the amount of enzyme required to digest 1 g of DNA (HindIII digest) fragments in 1 hour at 37C in a total reaction volume of 50 l. Concentration: 20,000 units/ml and 100,000 units/ml Unit Assay Substrate: DNA (HindIII digest) Storage Conditions: 10 mM Tris-HCl 50 mM KCl 1 mM Dithiothreitol 0.1 mM EDTA 200 g/ml BSA 50% Glycerol pH 7.4 @ 25C Storage Temperature: -20C Diluent Compatibility: Diluent A

Activity in NEBuffers: NEBuffer 1: 100% NEBuffer 2: 50% NEBuffer 3: 10% NEBuffer 4: 100%

When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion.

Methylation Sensitivity:

dam methylation: Not sensitive dcm methylation: Not sensitive CpG methylation: Not sensitive More information about: Methylation Sensitivity Heat Inactivation: 65C for 20 minutes Survival in a Reaction: (+ + +) Suitable for an extended or overnight digestion. Enzyme is active > 8 hours. More information about: Extended Digests with Restriction Enzymes

Reaction & Storage Conditions

Reaction Conditions: 1X NEBuffer 1 Supplemented with 100 g/ml Bovine Serum Albumin Incubate at 37C. 1X NEBuffer 1: 10 mM Bis-Tris-Propane-HCl 10 mM MgCl2 1 mM Dithiothreitol pH 7.0 @ 25C

Unit Definition: One unit is defined as the amount of enzyme required to digest 1 g of DNA (HindIII digest) in 1 hour at 37C in a total reaction volume of 50 l. Concentration: 20,000 units/ml and 100,000 units/ml Unit Assay Substrate: DNA (HindIII digest) Storage Conditions: 10 mM Tris-HCl 100 mM NaCl 1 mM Dithiothreitol 0.1 mM EDTA 200 g/ml BSA 50% Glycerol pH 7.4 @ 25C Storage Temperature: -20C Diluent Compatibility: Diluent A