Hello everyone,
I have had two same clones by using different cDNA from different cells. Before the sequencing, I got single clone of DH5-a bacteria and have PCR, restriction enzyme to confirm the positive. But the sequencing of the two clones had mutation at the end of whole sequence, especially from which I designed the my PCR clone primer. This is common of two plasmid. This is puzzled me. PS: sequecing primers are pcDNA3.1 CMV and BGH.
Could someone give me advice? Much thanks.
Hi Miao, first thing to check is that if the mutation will change the amino acid in your protein sequence. If not, continuing with your transfection or any other steps in your plan. If the mutation will interfere with your protein expression (by changing amino acid or resulting in stop codon), then you probably need to remake your primer (perhalps pick a different region to make primer if possible). When you confirm with PCR and restriction enzyme for the identity of the clones, it will not detect your mutation unless the restriction enzyme is cutting at the mutation site. Hope this helps. Good luck!
Hello,
Can you explain your problem in detail??
I don't understand if you have a double peak or a change in you sequence. Is it the same mutation/double peak in the two clones?
Are these changes exactly in you reverse primer sequence?? Maybe the primer is wrong if the sequencing results in the same change, after independent PCR with independent templates and separate cloning.
Hi Miao,
If your PCR primers are your sequencing primers, you need to know that due to the limitation of current ABI sequencing technology, the 'reads' of bases near the primers are usually not good. So, it will be better for you to sequence it in two directions (one from 5' end and one from 3' end) and sequence a couple of times. The resulting assembled sequence can then be used for analysis with a greater confidence.
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