sequencing issue - Internal primers (Jul/17/2007 )
Hi,
I want to sequence a fragment 2000bp long, it's in pGemt so I have universal primers on both sides......but as it's too long I need to order internal new primers.
Does anyone know if they can have sites for restriction enzymes on 5' side of the primers?
They would be useful for doing PCR afterwards so, if it doesn't affect sequenciation, I should order them with RE sites
How long should them be?
thank you!
You can add any kind of sequence feature to the 5' end of a primer. Anything from one or more restriction sites, to kozak sequence, stop codons, to self cleaving peptide sequence, to transcription factor binding sequence..
How long should them be?
It shouldn't be a problem adding the RE sites to the sequencing primer. However do remember to add sufficient bp around the restriction site, for the enzyme to bind to the site and cut. Also watch out for hair pin structures.
The lenght of the primer will be depend on its tm. For a sequencing primer you should aim for a tm of a round 58 Celsius. The tm of the primer is the tm of the sequence that actually binds to the template, not the entire primer.
Thank you pernesblue,
I'll keep that in mind, so I just have to take into account all the things to consider when designing any ordinary primer..........there is nothing misterious 
or special about the sequencing process....
bye
one thing though, the annealing temperature of the sequencing primer has to be below 60 Celsius (the extention temperature that the sequencing reaction uses)
just keep in mind that sometimes cloning primers don't work efficiently when sequencing.
Really, why not?
Where is the difference?
I still dont get it why people still want to use cloning primer to go for sequencing? Any particular reason?
I need to save money and as I'm studying a promoter sequence, I need to clone lots of fragments of different size....as I will probably need to amplified a sequence with the same primers I'll use for sequencing.............I just wondered if there was any problem in adding Restriction Enzymes sites on the edges of the primers I'm designing..
that's simply all........
Really, why not?
Where is the difference?
Hard to say, although one can speculate that the segment of the primer which doesn't bind to the template causes somekind of annealing problem... perhaps hair pin formation or non-specific template binding. Thus far I have had only one of clonning primer that would not act as a sequencing primer, and another one that work imperfectly. 2 out of the dozen or so cloning primers that I have used for sequencing.
see also this post.