Hi,

The "wrong" sequence can come from couple reasons. 

You first might wanna check whether your gene of interest has some variation. The variation can also come from mutation in specific sample. So you can try to have new source of genomic DNA to clone it.

Second it can be caused by error introduced during PCR of the gene. I would suggest to use Phusion or Q5 from NEB since this polymerase has high fidelity. Forget about Taq polymerase for amplifying gene for cloning.

Last maybe you might want to double check that the deletion not occur on the low confidence base called region due to sequencing limit.

Godd luck!

 i made a new genomic DNA , but it did not work, cause i got same result of DNA sequencing. the target gene is around 1680bp

Q5 from NEB i have never tried. Did you use that one? Does it work well? 

the last u suggested me, but i do not understand . Sorry T.T 

i really appreciate your help 

Starting over using a high fidelity polymerase is definitely the way to go. Q5 and Phusion are both good, and work well in my experience.

There are PCR kits designed for performing insertions, deletions and substitutions but these are generally more expensive and not ideal for your situation. If you're using regular Taq, try buying something better.

thank so much. I will try with Q5 

if the change is pcr generated you and as you now know the sequence of the problem insert you could design a primer pair that only works on the correct sequence in the vector and colony pcr many more colonies to see if you can find some colonies with the correct sequence

 i had tried many times, and sequenced a lot of colonies, but no any colonies are correct . Do you think the problem occur from PCR or subsequently PCR product into vector? 

Hi,

If you have in the lab a mutagenesis kit you can try to design a primer set to amplify your plasmid without the 3 additional nucleotides. It works generally quite well. If you have the same nucleotides all the time in your cloning I will think that you are amplifying a variant or that one of your primer is not perfect. I don't know the T-ease vector but if you have excised something before to use it again this can be the residues remaining after your digestion.

Hope this help.

Marie

 A frameshift mutation is a genetic mutation caused by a deletion or insertion in a DNA sequence that shifts the way the sequence is read. A DNA sequence is a chain of many smaller molecules called nucleotides. DNA (or RNA) nucleotide sequences are read three nucleotides at a time in units called codons, and each codon corresponds to a specific amino acid or stop signal. During translation, the sequence of codons is read in order from the nucleotide sequence to synthesize a chain of amino acids and form a protein. Frameshift mutations arise when the normal sequence of codons is disrupted by the insertion or deletion of one or more nucleotides, provided that the number of nucleotides added or removed is not a multiple of three. For instance, if just one nucleotide is deleted from the sequence, then all of the codons including and after the mutation will have a disrupted reading frame. This can result in the incorporation of many incorrect amino acids into the protein. In contrast, if three nucleotides are inserted or deleted, there will be no shift in the codon reading frame; however, there will be either one extra or one missing amino acid in the final protein. Therefore, frameshift mutations result in abnormal protein products with an incorrect amino acid sequence that can be either longer or shorter than the normal protein.

Hi,

make sure to use highest quality nucleotides, a proof reading polymerase mixture (i.e. long PCR kit, such as Triple master from Eppendorf), and as few amplification cycles as possible to avoid errors (try using 22-25 cycles). It should be possible to get out  correct 1680 bp in some fraction of obtained clones.

You could in parallel try another genomic template as a control.

Alternatively, as has been suggested, use site directed mutagenesis (only a couple cycles of amplification from the known plasmid template) to correct one of the clones you already have sequenced.

Good luck

Dirk

Maybe you want to design a two step protocol. Amplify your regions by two segments, using a primer where you don't incorporate those three nucleotides. Do the same for the second regions and make sure your 3'end primers from the first segment and the 5'end primers of your second segment overlap at least 40bp to perform a second step of gap repair. After gap repair you will have a complete sequence without those annoying nucleotides. Use phusion or Q5 to avoid unexpected mutation of your fragments.