lets suppose I have introduced SDM at position 15 T>G and after transformation, I sent the sample for sequencing, the results after sequences show many other unwanted mutations too, when aligned with wild type. what should be done to avoid unwanted mutations?
Did you use a high fidelity polymerase while performing the SDM PCR? The unwanted mutations could also have been generated when PCR was done before sequencing.
yes sir, I have used high fidelity polymerase even then i get such results
Did you sequence the original plasmid? I myself got a plasmid full of mutations, when I started my current work. And there are many more such stories.
In that case - grow overnight culture -> streak to obtain single colony -> grow overnight culture -> isolate plasmid -> sequence
Save this second overnight culture as your reference culture and you will see what does it look like.
I have cloned a bit in the last year and I didn't see "many other" mutations. Yes, from time to time, I saw some mutations, especially, if there were multiple identical nucleotides (like 10 Ts in a row), but it was not so bad.
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