Can T4 DNA ligase be inactivated?

T4 DNA ligase can be inactivated by a 10 min incu- bation at +65°C. Heat inactivation should be done if the ligation reac- tion mixture is used in experiments other than trans- formation assays. Otherwise a drastic decrease of transformed colonies is possible (factor >20).

Is T4 DNA ligase stable at high temperature?

The activity of T4 DNA ligase increases with an increase in the temperature up to its optimal temperature (37 °C). However, higher temperatures dissociate DNA fragments joined by base pairing at their overhanging ends, which decreases the ligation efficiency.

At what temperature is T4 DNA ligase most active?

Temperature. Temperature optimum of the most commonly using T4 DNA ligase is around 37˚С. Therefore, the best choice for blunt-ended DNA fragments is 37˚С. However, sticky ends are often too short to form stable duplex at this conditions; in that case ligation at lower temperature (4˚С) is preferable.

Does T4 DNA ligase require ATP?

T4 Ligase forms a phosphodiester bond between juxtaposed 5′ phosphate and 3′ hydroxyl termini in duplex DNA. To perform this catalytic reaction, ligase needs ATP. In the absence of ATP, phosphodiester bond won’t form and two ends of DNA won’t hold. ATP is essential for Ligase reaction.

How do you store a ligation?

Ligations can be done at room temperature or cooler (think 12-16°C) overnight or even for a few days, if you’re really busy. You can also store a ligation in the fridge and take it out later to continue ligating at room temperature for as long as necessary.

Do you need to heat inactivate T4 DNA Ligase?

Yes, heat at 65 °C for 10 minutes. Do not heat inactivate if there is PEG in the reaction buffer (Quick Ligation Kit buffer NEB# M2200 ) because transformation will be inhibited.

How do you inactivate T4 ligase?

Yes, T4 DNA Ligase can be heat inactivated by incubating at 65°C for 10 minutes. If the reaction buffer contains PEG, heat inactivation will inhibit transformation. In most common applications, heat inactivation prior to transformation is not necessary and should be avoided when possible.

How long does T4 DNA Ligase take?

Typical ligation reactions use 100–200ng of vector DNA. 2. Incubate the reaction at: room temperature for 3 hours, or 4°C overnight, or 15°C for 4–18 hours.

How can ligation efficiency be improved?

In general, increased reaction time, lowered reaction temperature, and molecular crowding all yield more complete ligation reactions. Reaction times can be increased as long as 24 hours or more. For ligations longer than 2 hours, we routinely incubate below 16°C.

How can I check my ligase activity?

You may check the efficiency of your ligation reaction by mixing the reaction with loading dye containing SDS (final SDS concentration 0,2%) and running it on a standard agarose gel. After ligation several high molecular weight bands should appear.

How long can ligation be stored?

It is always good to store at 4C for 12-36 hours and for long term storage (without repeated freeze and thaw) use -20C.

How long does DNA gel last?

Gel extraction of DNA from an agarose gel can be put off indefinitely. Try storing the gel slice in the fridge overnight, or even melting the slice in buffer and freezing it at -20°C or -80°C.