Cloning 180bp DNA in a vector
Haviland, David L
(by David.L.Haviland from uth.tmc.edu)
Sat Dec 18 17:49:31 EST 2010
In times past, I made riboprobes like that all the time. Planned for 200bp probe expecting to see 100bp by analysis on a gel. I used bluescript as I usually planned on blue/white colony selection, usually used 30 mers, cut, then ligated the PCR fragment. Used asymetic enzymes like BamHI and HindIII if I could. Trough eluted the vector, and probe. Picked colonies, worked them up, tried to see differences in single cut vector vs. one with the probe. And did a lot of blind faith sequencing. Between not using the same enzyme and blue/white color selection, sequencing ID' clones with regularity. We had an oligo machine in the lab and we never purified the products, we just lypholyzed and went many times on blind faith that the machine worked.
Analytical gel worked better if the probe was more like 400bp but I did have to make 200 bps at times.
Hope this helps.
From: methods-bounces from oat.bio.indiana.edu [methods-bounces from oat.bio.indiana.edu] On Behalf Of Pepa Florez Pérez [snipeurope from hotmail.com]
Sent: Saturday, December 18, 2010 15:29
Subject: Cloning 180bp DNA in a vector
I must clone a 180 bp. DNA that I have designed in several vectors.
Since it does not belong to a gene or cDNA I am not sure which one is the best method to carry out this project.
I have thought of two possibilities:
- to ask for 6 oligonucleotides, do the annealing and try to ligate them with a digested vector.
- to get the DNA synthesized by a company, and subcloning by digestion.
The problem is that I don't know which one is the proper approach, if there is any.
About the oligos, we talked about not asking them purified by HPLC or PAGE since these are quite expensive procedures, but I am afraid that becuase of that I won't have a good annealing.
And about the DNA synthesis... it cost around 200 euros/each... what we have considered as quite expensive too.
Do you think that we will be successful working with not-purified oligos? Or something cheaper will become expensive late?
Can you think of any other alternative?
Thank you very much
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