please tell me more about primers with restriction site! - (Aug/28/2006 )

ok i feel like a complete idiot. i mean i dont even try to compare myself with my genius freind here (who is doing her phD also) but every time she makes an argument i feel like . anyway this is the story:

i make primers with RE sites attached and some clamp sequences and make my insert by PCR. in this case my primers are not similar completly to DNA template of course. i make ligation etc and i get colonies and try to check by PCR and i fail. my professor says it is because my primers are not similar with the sequence of DNA. so im like ok ok ok i see. and now i tell that to my freind and she say what??? but you constructed the insert with those primers and they were even less similar than now (RE included now) and it worked. and again i am lost.
why is it always that insert construction works so great and if you back to checking stuff you constructed it's all failure. is there any difference? please please explain to me

hi kathy

i don't understand the problem. if you have got a pcr product in good amounts then your primer sequence is built in the pcr amplified product, and if these primers worked once to give you the product they will work again when you are screening your colonies. i don't think there should be a problem here.

if you not getting your product in the colonies you are screening, how are you sure that the insert is there after all.

you could do any one of two things easily:

1. do a miniprep and digest the plasmid with the restriction sites you introduced. if your product is there, you will see it excised out on the gel.

2. you screen your colonies by pcr with the forward and reverse primers flanking the mcs in your vector. in case your insert is there, you will see a band that would be of a size "your fragment+mcs", as against just a small fragment if your product is not there.

good luck

- viv

thanx a lot viv, yes i did miniprep and i will going to repeat it, since i am not getting much DNA to check the plasmid. and i can't do pcr with vector primers since we dont have them here.
about my question im really lost please can you clarify further, so what you mean is that even if half of the primer sequence is not complementary to the template DNA it should multiply it. since it already did once. right?

just understand one thing kathy, that your pcr product is "primer to primer".

to begin with, in the first two cycles, your primer may not have had complete homolgy with the template and may have hybridized with the template on the basis of partial complementarity, however, third cycle onwards, you will get a product which would be from the 5' end of your forward primer to the 5' end of your reverse primer. you could just draw the first three cycles on a piece of paper and you will understand what i mean.

the fact is that you have got the product, which means that the partially complimentary primer has worked.

since you must have introduced restriction sites on the 5' end of the primer, and since you have used those restriction sites to ligate into vector, it is clear that the restriction sites exist on the 5' end. if they do, then obviously the remaining part of the primer sequence is also in the pcr product, and hence, if the cloning is right, then when you use the same primers on your colony plasmid, it should give you the product as produced by the initial pcr.

hope i am clearer this time. assuming of course that i have understood your queries correctly.

- viv

thanx a lot viv! yes i understand now. what my professor has said has confused me, but of course i should be able to detect my insert with same set of primers. have a great day!