I have a insert in my vector.
The size of my insert is approximately 6.7 kbp.
When I sequenced the insert, I found 3 error sequences.
I want to replace the error sequences with correct sequences.
I tried to replace the errors using site-directed mutagenesis method,
but that resulted in forming other error sequences in different region.
I have tried restirction/ligation method, and I have got to replace
a error sequence with a correct sequence using BamH1 enzyme,
but it is too difficult to find out appropriate restriction enzyme sites.
How can I easily replace error sequences in my vector ?
Maybe you could amplifie your whole insert again and put it into a vector?
I think that would be easier than to replace three different regions of your insert - as you say, finding restriction sites would be difficult.
If it is indeed in the vector why do you care?
If it is indeed such an issue Sophie Ziegler response make a lot of sense... Just re-clone
Yoram
Are you sure they are 'errors' and not just SNPs?
What is the source of your insert? another plasmid or cDNA or something else?
1. What DNA Taq polymerase did you use to amplify? If you re-do, use a High-Fidelity DNA polymerase for your PCR.
2. you should re-sequence those 'error' bases, to make sure that those are real, not just some sequencing errors.
Sophie Ziegler, Yoram Gerchman : I think that if I re-amplify my insrt, it will result in forming other error sequences in different region.
Maximilian Peters : I am not sure they are 'erroors'. should I make another fragement to make sure they are error not just SNPs ? The sourse of my insert is genomic DNA of a plant pathogenic fungus.
Yuan-Yeu Yau : I wil re-seqeunce the error sequences
if you use a high-fidelity DNA polymerse you should not have problems with errors - I did a lot of cloning and I can't remember re-doing something because of that
You can clone your insert using homologous recombination. It can be done in the E.coli or yeasts. You can amplify your sequence as several fragments with overlapping ends. The fragment on both ends of your insert must overlap with the vector sequences. The length of reliable overlapping that guarantees correct assembly is about 30 bp for E.coli and 40 bp for yeast. Number of fragments may be about 10 or more. The lesser fragment the lesser probability that it will have a mistake during PCR. Use your linearized vector and purified PCR products directly in transformation of yeast or E.coli cells. E.coli cells should be recA+ strain. Then look for colonies with full-length inserts. Plasmid from yeast can be purified with Quagen plasmid purification kit with minor changes on the lysis step. I have successfully assembled a 2 kB insert from 7 fragments to introduce 5 mutations. In your case you can fix 3 mutations with corresponding primers. See detailed protocols for E.coli in the files attached.
Good luck!
Thank you everbody for the opinions.
I have start to amplify my seqeunces as several fragments overlapped at each end.
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