I have designed stem loop primers with last 6 nucleotide of gene of my interest and respective forward primers and universal reverse primers against stem loop primers. I got successful cloning and after sequencing i found my insert containing target sequence but last 6 nucleotide ( which was part of stem loop ) was missing .
This happens, look for other clones. Synthetic oligos are made in the 3′−5′ direction, you could have in your preparation shorter interediate of syntesis. Buy primers HPLC purified, they are >85% full-lenght (thermofisher scientific).
This happens, look for other clones. Synthetic oligos are made in the 3′−5′ direction, you could have in your preparation shorter interediate of syntesis. Buy primers HPLC purified, they are >85% full-lenght (thermofisher scientific).
Which primers u used for sequencing? Have u done forward and reverse sequencing? Use universal primer for sequencing. If u have done sequencing with your own primer then there may be missing in reading of initial sequences. So first do the forward and reverse sequencing if using your own primers.
Thanks Dear Paola. My primers are HPLC purified . Though i will repeat this again and also for other clones also.
Thanks Amit Singh. Actually i did forward and reverse , both side sequencing with T7 and SP6 primers , which are specific for PGEMT vector. .
Dear paola, yes it might happen . Need to repeat experiment.
Many suggestions to avoid this situation.
Increase the annealing Temperature and if it is a GC-rich sequence you could add some buffer constituent ....DMSO, betaine . I would order a new primer adding a restrction enzyme to use during clone screeningArticle Betaine, Dimethyl Sulfoxide, and 7-Deaza-dGTP, a Powerful Mi...
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