cloning primer and tag questions - first try (Jun/29/2006 )
I am about to dive into my first try at cloning into a pYES2 vector. I am considering sticking an HA or MYC tag on the construct (since there isn't any in the vector). As I understand it I need to put the tag sequence on my primers, but this will make them 60bp long....is that okay? are there any wierd rules for cloning primers (besides RE site and being sure the sequence is 'in frame')? I also read about a group putting 3 HA tags on their PCR fragment using primers....(about 120bp), does anyone have any experiences doing this? Lastly, if I'm just looking for presence or absence of protein would I need multiple tags, or would 1 tag be suffice?
I have used a single HA tag and it works well to detect the protein. Confirm where u want the tag to be, N- terminus or C terminus. U will have to design a primer approx. 60bps, its works.
hey, Eric. I totally agree, but I would add one thing. a long primer is not a problem, but make sure to be careful with the Tm of the second primer...and consider also the Tm of the complementary portion of the large primer vs the smaller primer. if the discrepancy is too great you will need to allow for this when programming your thermal cycler. perhaps if you have time you might flip through some of the older posts here on this forum using the search engine; I recommend "Tm" and "primer annealing" and such as keywords...you may have to wade through a lot of stuff but there should be some good info on how to allow for the Tm discrepancy. Of course, if you get stuck you can always ask us