I have 637bp double-stranded DNA with 5’ end digested with BamHI / 3’ end digested with SacI.
I need to ligate end-specific adapters to each end.
The adapters consist of 17bp double stranded DNA with complementary cohesive ends with a 55base single stranded 5’ overhang.
I will end up with 671bp double-stranded DNA fragment bearing 55bp 5’ overhangs at each end.
I will therefore not be able to transform bacteria with this fragment and bulk-up in the normal way so I need optimum ligation conditions to ensure I get a sufficient yield of product at the end.
I must have missed something... Are you going to clone your fragment in a plasmid? Does the plasmid have complementary overhangs? Why do you need these 55 p overhangs?
estanis
No plasmid involved. Just need my gene of interest with 55base 5' overhangs to insert directly into the genome of my cell line using CRISPR. Cas9 double nicking will provide complementary overhangs in the genome exactly where I want to insert my gene.
To ligate the adapter to the fragment I would try to first anneal the construct and then add T4 ligase. Mix fragment, 5' adapter and 3' adapter in a minimum volume (25 ul), heat the mix at 65-70ºC in a water bath for 10 min and then let cool down very slowly (turning the bath off will do it). When the water is at room temp add the ligase, buffer etc. I think that you will have to work out the conditions (amount of adapters, ligation time...) to improve the yield. Should not be a difficult ligation but, depending on the efficiency of the reaction you can end with an excess of unligated adaptors ...
hope this helps
estanis
Agata, I know the sequence. I have PCR amplified my gene but then I need to attach 55 base single stranded overhangs to each end. This will be complementary sequence to a single strand overhang in the genome I have created. I annealed two oligos to get the overhangs - 68 bases top strand and 13 bases for bottom strand to give double stranded DNA at 3' end to allow for ligation to the double stranded PCR amplified gene. I also have a pair of oligos annealed with double stranded DNA at the 5' end to ligate to the 3' end of my gene.
For optimal ligation efficiency perform a minimum 5:1 molar ratio adapter:DNA reaction and clean up with Ampure beads (or other magnetic bead system) to prevent insertion of unligated adapters downstream. Quantify DNA molarity on BioAnalyser (or calculate from Nanodrop concentration). T4 ligase should be sufficient, you can add extra ATP to a final concentration of 2 mM but this shouldn't be necessary.
Hello, as I follow your question, you can design your primers with your long adapter follow by your primer sequence to amplify by PCR your gene of interest. The primers will be pretty long, but your Tm° will be the one that binds to your gene. I
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