2 replies to this topic

[subtelo]

Hello all,

I have an around 4k bps DNA fragment. It comes from restriction digestion.  I want to fragment it in several small pieces and clone them into an vector and then do sequencing. This 4k sequence data is unknown, and it is failed with PCR amplification. I have 2 questions regarding the experiment. The first one is which strategy I can use for cut the 4k DNA into a couple of small fragments, the best choice is 500 bps for each fragment. The second question is that which cloning kit I can use to cloning these small fragments.

Any recommendation will be highly appreciated.

Edited by subtelo, 07 December 2010 - 09:38 AM.

[NBaye]

Hi,
From what I gather, you have no idea as to the sequence of this 4kbp fragment?  If that is the case then I would take the fragment and see if I could snap it into something like pRSET or pETTOPO from Invitrogen (personally, I've just had a lot of experience using these vectors) using the restriction sites that you already have at the ends of the fragment if possible or you could blunt clone it in by filling in the ends of the fragment and using an EcoRV or PvuII site in the vector.  Then, I'd test the insertion by re-cutting, if possible.  Finally, I'd send for sequencing to someplace like Davis (www.davissequencing.com) where they have inhouse primers for the T7for and T7rev which flank the MCS of both of the above vectors.  From there I would keep sequencing through the insert by developing primers from the sequence gathered.  That's about all I've got.  Hope all goes well!
~Nic~

[subtelo]

NBaye, Thanks for your recommendation. I am not familiar with pRSET and pETTOPO. I will dig into a little bit and see if it meets what I want to do. I am still think I need cut the fragment first since I already failed to sequencing through the fragment. All primers that I can find are stop at the same spot. Thanks again.