I cloned 1.6 kb gene into pTAC2 expression vector (linear : 2786 bp). I performed a diagnostic restriction digestion to confirm the insert. I used Bigdye terminal v3.1/1.1 cycle sequencing kit and Hi-Di Formamide. Plasmid DNA concentration was about 100 ng/ul (1 µl) and primer was 1 pmol (same primers that I used in sub-cloning time) in sequencing reaction.
Run PCR: 96C for 1', [96 C for 10", 50 C for 5" and 60 C for 4'] x 25, 16 C forever.
Then, DNA was precipitated by ethanol (100% and 70%) with 125mEDTA. After that, i added Hi-Di Formamide and keep 95 C for 2 minutes. Then, I did sequencing and got noisy electropherogram peaks ( Please see attachment). Any suggestions will help me a lot.
Thank You !
I think that you need 3.2 pM primer for sequencing. your sequence is just a bit weak compared with the background so more primer and even a couple more cycles. Check the size of the vector, you need about 20-40 ng of insert and the insert is only a fraction of the total dna ( the rest is the plasmid). If you could attach an actual .abi file that would give much more information than a picture of the electrpherogram
You may need to linearize your plasmid before sequencing. Or PCR amplify your region of interest and sequence the PCR product.
I would sequence 3-5 times more template and with more primer as above. Also when you have precipitated the sequenced dna I would wash again with 70% ethanol to remove residual salts before dissolving for loading. Capillary sequencing loads by electrokinetic injection so if there are too many salt ions around they load preferentially and very little sample loads so you get a weak signal
Dear Katie A Clark
Thank you so much for your suggestion. But i did PCR amplify of my region of interest . and finally failed the sequencing .
Dear Paul Rutland
Thank you so much for your suggestion. I will follow that. In addition, I washed firstly by 100% and then 70% ethanol. Thank you !
Hi, I think that there are two steps for this experiment including the PCR step and the sequencing step. Firstly you should check whether you obtained the right PCR amplicon via running a normal agrose gel. If the size of the amplicon is right and there is only one unique band, then you could go to the sequencing step. Otherwise you should optimize your PCR mixes or program. In the sequencing step, you could follow the suggestions from Paul.
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