I am having a problem with the sequencing of recombinant plasmids. I need to get sequences in both forward and reverse directions. For sequencing, I am using M13 Forward and Reverse primers separately. In 99% of cases, I get good sequences with the reverse primer. The sequences correspond to the expected size of my gene (approx 1000 b.p.). But when I try sequencing in forward direction I cannot get a full sequence. The fragments are approximately 150 b.p. I have attached the example of sequences that I received. Could anybody help with this?
Thank you in advance
I am having a problem with the sequencing of recombinant plasmids. I need to get sequences in both forward and reverse directions. For sequencing, I am using M13 Forward and Reverse primers separately. In 99% of cases, I get good sequences with the reverse primer. The sequences correspond to the expected size of my gene (approx 1000 b.p.). But when I try sequencing in forward direction I cannot get a full sequence. The fragments are approximately 150 b.p. I have attached the example of sequences that I received. Could anybody help with this?
If we make a contig of the 2 sequences M13r gives a weak intensity but very good clean and well spaced sequence from about 200-890 ( contig base numbers) getting much weaker in the bases below 200. This is normal sequencing and template and primer are correctly matched in concentrations. With M13F the sequence is very strong in intensity from 80-230 of the contig dropping off very quickly to give no analysable signal after base 230. This usually indicates too much template or too much sequencing primer in the sequencing reaction. Since M13R worked well we might assume that the amount of template is the same in both reactions and is correct so my guess is that you have too much M13F primer giving a very strong early signal but depleting the sequencing mix completely so you cannot get any long sequence. The solution is either use more sequencing reagent mix or less M13F primer
If we make a contig of the 2 sequences M13r gives a weak intensity but very good clean and well spaced sequence from about 200-890 ( contig base numbers) getting much weaker in the bases below 200. This is normal sequencing and template and primer are correctly matched in concentrations. With M13F the sequence is very strong in intensity from 80-230 of the contig dropping off very quickly to give no analysable signal after base 230. This usually indicates too much template or too much sequencing primer in the sequencing reaction. Since M13R worked well we might assume that the amount of template is the same in both reactions and is correct so my guess is that you have too much M13F primer giving a very strong early signal but depleting the sequencing mix completely so you cannot get any long sequence. The solution is either use more sequencing reagent mix or less M13F primer
- Badges
- Science method