I am using two inverse PCR primers to add a sequence into my plasmid. After PCR I am phosphorylating and ligating it. Also, I am using bacterial transformation to antibiotics resistance check of the plasmid. After all, I am using another primer pairs covering the sequence added region to check the size in colony PCR from survived colony samples of the plate. Afterward, I am sending the best PCR results after PCR purification to sequencing. However, half of the sequence, which is in one of my primers as a single strand, I am trying to insert is missing in the sequencing result. Can it be broken in the tube because of vortexing or something? If I dilute my main stock again and repeat the experiment will it work? This is the only idea I have, I am open to any suggestions. Thank you so much!
I may be misunderstanding the problem here but always when you sequence the sequencing primer and the next 30-40 bases are unreadable then you get good sequence for up to 900 bases, If you use the reverse primer for sequencing then you do not get that primer sequence or the next 30 bases but the sequence from the reverse primer will include the forwards primer . Normally in sequencing you only see the sequence of the other (non sequencing) primer.This is no problem if you sequence in both directions but if you only sequence in one direction you can never see the sequencing primer because the sequence starts at the base following the 3' end of the primer
Mustafa Divyapıcıgil I agree with the comments from Paul Rutland. Sequencing from both the ends is important if you are using specific primers from insert ends. Alternatively, you can can use primers in the plasmid that is at least 70-100bp from the insert ends to get good quality reads!
You can use a sequencing primer from your promoter and terminator regions. Probably your vector has a common forward and reverse sequencing primer pairs such as T7 forward primer etc. Then you will receive the complete sequence of your clone.
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