cloning large fragments
pullabhotla sarma
Monday, 13 August 2007 11:35 UTC
I want to clone large DNA fragments isolated from soil, i am following general cloning method of sambrook (molecular cloning )using calcium chloride method for transformation.I am facing problems with ligation where i could not able to ligate more than 1-1-2 kb. Kindly suggest me some tips and other protocls which can help me to clone above 5- 10 kb.
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Replies
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hi i think u can have two ways
1. you can try some vectors which can ligate larger fragments (e.g.YAC).
2. The other fast way u can clone ur desired oligos is through PCR. Make the primers and go for amplification. after this u cut the DNA bands from the agarose gel after running gel and purify using commercially available kits. -
May I also suggest Invitrogen’s Gateway cloning system. This has worked very nice for us, also for cloning large fragments. Though we never tried >5 Kb.
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well yac is a little bit unstable over time isn’t it? cosmids should do better for only"5-10kb cloning…
Maybe PCR the vector too should help to get unphosphorylated form.
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