Dear All
I need to make single stranded DNA fragments. I see that you can biotynilate one of the strand with a biotynilated primer and then separate this specifically with streptavidin-dyna beads. I would like to have a detailed protocol or if there is any other possible way of doing it.
Thank you
Ikram
If you use Chelex for the extraction you will get single stranded DNA
Classically, heat denaturation (96oC 10 mins then fast cool in ice-water slurry).
pH greater than 11
8M urea.
Yes, one sided PCR, which isn't PCR, of course as its linear amplification.
Commercial synthesis.
NOT biotinylation unless you use a strategy that only labels one strand and not both strands. So you could PCR using two primers, only one of which is biotinylated. You can remove the biotinylated product using beads (+8M urea, or heat denaturation) or even strep coated microtitre plates. You just need to check the capacity needed.
It really depends on what you will do with it.
I think Jun Song's answer assumes that you are reamplifying purified pcr product and is correct. Asymmetric pcr using both primers but one primer in large excess and many (up t0 50 ) cycles can also be used from genomic dna so the correct size product is amplified and then linear amplification enriches the strand that you want
Dear Jun and Geoff
Thank you for your answer. However, I am a bit skeptical of doing a PCR with just one primer, I am not very sure whether it will work, however, I will still give it a try because that sounds the easiest. And, yah, I would use a PCR product as the template.
Dear Geoff
At the same time I ordered a Primer, only Forward primer with biotibylation at the 5'-end. The other primer will be the normal one. Hence, only one strand should be biotinylated. We have streptavidin beads that I could use to separate the biotinylated strand. Thank you for suggesting the high temperature or alkaline conditions for the separation.
Lets see how it works on Monday.
Thank you very much.
Ikram
I am certain that Jun's suggestion of linear amplification will work, but the amount of material you will obtain, as Jun says, with 30 cycles is only 30 times the starting material. With PCR proper (two primers) 30 cycles will theoretically give you about 1,000,000,000 times the starting material. Correct me if I'm wrong, Paul!
accurate Geoff 2exp10 is nearly approximately 1000 so every 10 cycles of pcr gives 1000 times more product in an exponential amplification but single strand cycling is only additive ( linear) so just adds the same amount of single strand with each cycle . Of course with amplified product to start with there will probably be quite a lot of starting material so 30 times that will still be quite a lot and about 97% will be single strand. Depending on what it is needed for it might be relevant to consider which enzymes add an A base and which do not just in case an extra A makes any difference to the next stage
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