DNA Ligation Troubleshooting

DNA Ligation Troubleshooting

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Molecular Cloning Strategies

What is PCR

Restriction Enzymes Digestion

DNA Ligation

Plasmid Transformation

DNA Transfection Guide

Transfection Protocol

DNA Transfection Troubleshooting

Ligation Protocol

DNA Ligation Troubleshooting Guide

PCR Protocol, PCR Steps

PCR Troubleshooting Guide

Restriction Digest Protocol

Restriction Digestion Troubleshooting Guide

DNA Ligation Troubleshooting

What is DNA Transformation|Biology

DNA Transformation Protocol

Transformation Troubleshooting Guide

Thaw all reagents on ice.

Assemble reaction mix into 10 µL volume in a microfuge tube. Reaction may be scaled up to 20 µL if DNA concentrations are low.

Add reagents in following order: water, buffer, insert, vector, T4 ligase.

Gently mix by stirring gently with pipette tip.

Typical Incubation time and temperature is 15°C for at least 4 hours. Incubation at 4°C for overnight is commonly used.

Prepare a control that excludes the insert. This allows for detection of re-annealing due to incomplete vector digestion (If vector is not completely digested the colonies generated by the vector reaction will greatly outnumber that of the vector + insert reaction).

Ligation Reaction can be inactivate d by incubation at 65°C for 20 minutes. This step is optional.

Proceed directly to transformation reaction.

Molecular cloning strategies

PCR

Restriction digestion

Ligation

Transformation

Transfection

Molecular cloning handbook

Bioinformatics tools

water

to 10 µL

Ligase buffer (with ATP)

1X

Vector

25 ng

Insert

75 ng

T4 DNA ligase

0.1 to 1 Weiss unit

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