Ligation and cloning failure - (Jul/30/2006 )

Sir, I am Going to Die I don't know what to do.

Since last year I am trying to ligate one cDNA and two gene amplfied from Genomic DNA into P1301s (Modified from P1301). Vector is 9kb

1,the cDNA genes is 800bp, RE BamH1 and Sal1
2,Gene amplified from Genomic DNA by PCR 2.1KB RE Kpn1 Sac 1
3,Gene amplified from Genomic DNA by PCR 3.6KB RE Kpn1 Sac 1

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Have you checked if the restriction enzyme sequences are present in your gene sequences?

Also, after PCR of my DNA I used to put the DNA into TOPO vector as a holding stage (TOPO vector's multiple cloning site I believe has all of the RE you are interested in). I then digested the DNA out of the TOPO vector along with digesting the vector the DNA was going into, and then ligated.

I actually ended up subcloning for over a year to get my constructs (frustrating!!!) thru the same process of PCR digestion and ligations....my major problems were RE sequences in my DNA sequence and my reverse primers for PCR were incorrect.....sometimes just the basics are the root of the problems

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Thanks Mimini,
My primers contain the RE sites and also I used TA cloning system Promega. Sir do u think I have to sequence my purified PCR product. Please give me a detailed solution.

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u have to sequence what ever u have PCR amplified.

Also take care while u digest with SalI. I had some problems with this RE.

I am not sure abt SacI, havn't used it.

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