help design primer - (Sep/19/2008 )
I want to design primers for the promoter (I wish to fuse my promoter with GUS reporter gene)
I need smaI restriction site (CCC’GGG)
here is the promotor just in case anyone can see an obvious primer combo
appreciate your help
Design primer and post it and we will check if its fine.
appreciate your response
forward: 5' AATCCCGGGCGTGTGGATGCC 3'
Reverse: 5' gccccggggaaaaagcaagaag 3'
i did not get a right amplification (size) plus lots of non specific bands
how much wiggle room do you have? Is the ~1.8 kb the entire promoter? Where is the promoter exactly. And where is the SmaI site going to be located, 5' of the promoter or 3'? and what is the other restriction site
I agree with scolix, you should design the primer first and let us check it. You know best what is to be done with this fragment and the overall ligation strategy.
But to help things along.
Forward primer tm = 60
GTA AAC AAT ACA CGC CGT GTG
Reverse primer tm = 61
warning (contains BamHI restriction site).
GA AAC TGG ATC CGA CTA ATC AAC
GTT GAT TAG TCG GAT CCA GTT TC (sequence reverse primer binds too)
More work need be done. But assuming the entire sequence here is the promoter, I think you could use these guys as a starting point. Note the BamHI site that is present on 3' end of the template. Is this good, bad?
Thanks for the primers, lets have a look here......
Guard : AAT
SmaI : CCCGGG
Binding sequence : CGTGTGGATGCC tm = 40 Celsius
AAT CCCGGG CGTGTGGATGCC
Umm... the melting temperature of this primer is 40 Celsius. I am sorry to say but this primer can not be used for cloning work. It also has hairpin loop formation problems.
Guard : GC
SmaI : CCCGGG
Binding sequence : gaaaaagcaagaag (reverse complement) tm = 35 Celsius
GC CCCGGG gaaaaagcaagaag
Umm... tm is 35 Celsius. Guard is too short.. possible problems with SmaI digest.
Mine if I inquire to the ligation strategy? Blunt end ligation is not the easiest thing to do. And with a blunt end on both ends of this fragment, you will face problems from concatemers and multiple inserts into the vector. Is it at all possible to modify the ligation strategy and use sticky end restriction sites?
useful site to visit are
Northwestern oligo calculator very useful to determine tm of the primer and hairpin structure
NEB technical reference, useful for looking up restriction site sequences and the minimum number of bp required to skirt a restriction site for efficient digestion.
thanks for the lovely info,
i checked my primers with IDT and they seemed okey so i ordered,
i will recheck in your program
maybe i will have further queries to comeup with,
ah, I think I should clarify myself, the tm of a primer used for cloning work is based only segment of the primer that actually binds to the template. A primer may be 100bp long but if only 18bp actually binds to the template only these 18bp are used to calculate the melting temperature.
my bad i was actually misleading my self and now i realize what blunder i have made.
i was going back to my vector and i found that there still is NcoI on the 3' of the promotor that i can use
there are tons of other enzymes that could be used but the problem is some of them cut my promotor and others cut in other regions of the vector as well. and finally i dont want any of the promotor regions of the vector itself to be included in my construct.
so i am actually narrowed down to a small number of choices
and oh yes
may be i could use XmaI instead of smaI to generate 5'extension
finally thanks again for everything and i will come up with any other possiblity if i find any