How can I improve the ligation efficiency from DNA fragments extracted using the Gel DNA Extraction System?
Under certain conditions, enzymes from the ligation reaction may bind to NucleoPur particles affecting ligation efficiency. Centrifuge samples for 30 seconds and pipette the purified product into a new microcentrifuge tube.
Due to the chaotropic nature of the buffers used in the Gel DNA Extraction System some of the DNA may have denatured during the extraction process.
In order to promote renaturation of the single stranded DNA one can:
A. Elute the DNA from the NucleoPur column in 10mM Tris-EDTA buffer.
B. Set up the downstream reaction without the enzyme and incubate the mixture at 95C for 2 minutes to reanneal the single stranded DNA. Allow the mixture to cool to room temperature slowly before adding the enzyme and proceeding.
Due to the chaotropic nature of the buffers used in the Gel DNA Extraction System some of the DNA may have denatured during the extraction process.
In order to promote renaturation of the single stranded DNA one can:
A. Elute the DNA from the NucleoPur column in 10mM Tris-EDTA buffer.
B. Set up the downstream reaction without the enzyme and incubate the mixture at 95C for 2 minutes to reanneal the single stranded DNA. Allow the mixture to cool to room temperature slowly before adding the enzyme and proceeding.