Assume we are given an aliquot with many different double-stranded DNA fragments mostly of size 50 bp - 1000 bp. I would like to amplify all these fragments at once from end to end for each framgent.
A good way could be to use multiple displacement amplification (MDA). However, with the random hexamer primers I could also amplify "subfragments" of my fragments, which would be undesirable for my experiment.
Do you know a better way to achieve that goal here?
Also do you have a good protocol either for the better method or for MDA? And how high would the cost per amplification be?
Hi Tobias,
A simple way would be to ligate a single dsDNA adapter to both ends of the PCR products at once.There are some protocols online for this. The main issue is that neither PCR product nor regular oligonucleotides are 5' phosphorylated but there are ways to take care of that. Once the adapter ligated you just have to amplify everything with a single primer corresponding to the sequence of the adapter.
Hope this helps
Nick
Hi Tobias,
A simple way would be to ligate a single dsDNA adapter to both ends of the PCR products at once.There are some protocols online for this. The main issue is that neither PCR product nor regular oligonucleotides are 5' phosphorylated but there are ways to take care of that. Once the adapter ligated you just have to amplify everything with a single primer corresponding to the sequence of the adapter.
Hope this helps
Nick
That's the way I'd do it. Ligate a linker onto both ends and amplify.
Ligating adapter at both ends will be easiet way to achieve your goal. But what is your altimate aim after amplification is improtant to use downstream method of for analysis . Alternatively you can add the linkers with rare restriction enzyme site, clone it and sequence.
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