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Lambda DNA ligation

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#1 GK_N



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Posted 26 June 2011 - 10:11 PM


I have problems to ligate 12 bps oligonucleotide to the sticky end of Lambda DNA (48502 bps in 1xTE from NEB) using T4 DNA Ligase(NEB, 2,000,000 units/ml) . The oligo sequence is complimentary to one end of Lambda DNA.

Most of T4 DNA Ligase protocol online is based on vectors which is much smaller than Lambda and inserts which is larger than my oligos. So I don't know how much Lambda DNA, oligo and T4 ligase to use. I tried once using 0.5ul ligase(1000 units), 0.125pmol Lambda DNA (final c=0.07875ug/ul) and 0.75pmol oligo in a final volume of 50ul, incubating at 16 degrees for 24hours followed by isopropanol precipitation for purification. And it gave me a very low ligation (maybe less than 1%). So I just need some help about this issue.

Thank you very much. First post :)

#2 phage434



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Posted 27 June 2011 - 04:13 AM

Ligation of single stranded DNA to a sticky end double stranded piece of DNA does not occur (at least with T4 DNA ligase). You need to anneal your oligo with a complementary oligo to form the correct overhang double stranded short strand. This will likely be difficult with a 12 bp strand, but perhaps possible. Concentrations that are 2-3 molar high in the double stranded oligo are probably about right. This means very low amounts of your oligos compared to your lambda DNA.

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