If you do the primer sequencing, remember that the first bit of sequence data is not good enough to use and that any primer region will come back as an exact match to the primer. If you really need the entire sequence you'll need to clone your fragment into a vector.

Hello Sayanti Halder

Please refer to the article below.

Article Guidelines for Sanger sequencing and molecular assay monitoring

Highlights:

Suggestions for short sequences (< 100 bp)

Sequencing procedures are most efficient for amplicons ranging from 100 to 800 bp. Because many rtPCR assays are designed with amplicons of 65–100 bp, obtaining sequence results for rtPCR amplicons presents a challenge. Although sequences from amplicon < 100 bp can technically be generated, quality outcome for these shorter fragments cannot be guaranteed. Whether performed in-house or outsourced, those responsible for the equipment used in generating the sequence data should be alerted to all samples containing short amplicons (e.g., < 100 bp). Dependent on the equipment platform used, it is often possible to apply a sequence-analysis modification within the instrumentation software that will improve the outcome for short targets.

Other possibilities for sequencing short targets include the use of an “outer primer set” that amplifies a larger segment encompassing the specific target segment of interest. Because the primer sites are included, this option is also beneficial for monitoring PCR assays and for performing quality control trend analysis for evaluation of assay efficiency. It is additionally possible to clone the rtPCR amplicon into a vector, followed by sequencing using vector-specific primer sites (M13, SP6, T7, etc.). Some commercial sequencing facilities recommend specific vectors and maintain vector-specific primers in stock for their clients. Finally, M13, which is a common priming site, can provide an option for sequencing short amplicons. The M13 sequences can be tagged onto the original forward and reverse primers followed by a limited amplification (e.g., 15–20 cycles) performed with the tagged primers27 for sequencing short amplicons resulting from real-time assays. This technique is reported to effectively provide the sequence of small amplicons ~ 50% of the time.

I hope this helps.

Best.

Malcolm Nobre Okay, Thank you so much.