I'm trying to clone a E.coli 17 kb plasmid DNA for sequencing. I have contaced differenr company but their price is too high more than 55k.CAN anybody help me
1)how can i clone and sequence 17kb plasmid??
2) can anybody send me any vector that I can clone .
You can digest the plasmid with any common enzyme (say, EcoRI) and gel-purify a smallish (less than 500 bp) fragment, clone it into any general purpose vector (say, pUC or pBluescript) and send it for sequencing with universal primers. Once you get the results back, you can do primer walking on the original plasmid. Lengthy, but will set you back WAY less than 55k.
You can digest the plasmid with any common enzyme (say, EcoRI) and gel-purify a smallish (less than 500 bp) fragment, clone it into any general purpose vector (say, pUC or pBluescript) and send it for sequencing with universal primers. Once you get the results back, you can do primer walking on the original plasmid. Lengthy, but will set you back WAY less than 55k.
I am thinking that as long as we can bring a DNA fragment of known sequece into the unknown plasmid, then the sequencing can start from using the inserted fragment as a priming site. I don't know whether any such kit exists in the market.
Transposons have been used for 'generating sequecing priming sites' in plasmids (see attached papers). But I have not done this kind of experiments, so I am not sure how complex or how easy using this systme for sequencing unknown plasmids or long DNA fragments.
If the markets have such kits with a simple way to generate sequencing priming sites, then it will be very cool. One insertion, theoriotically, is enough to sequence the whole plasmid.
@ Sandip Kumar Mukherjee,
Why is so expesnive, 55K? 55k of what? US dollars?
For now, I think Primer Walking is a good way to go as usggested by Alejandro Martin. Slowly but surely!
Interesting! I do find this transposon kit in the market (see attachment). It called Primer Island® Transposition Kit - Applied Biosystems
It is from yeast Ty transposon, which is the only transposon can work in vitro (see the introduction). It can be used for inserting priming sites for bidirectional DNA template sequencing. The transposon can insert into diverse DNA, including plasmid.
Thank you Alejandro Martin and Yuan-Yeu Yau for your kind information.I will follow your suggestion.
I toiled the early part of my PhD subcloning bacteriophage inserts into plasmids for restriction mapping and sequencing. 20 kb inserts cloned into pBluescript are possible, but require patience. You need high efficiency electroporation and hope that the plasmids can propagate with rearrangement. Try different strains if you can't get the full length insert cloned.
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