no results in RACE PCR - (Feb/09/2007 )
it is my first time to do RACE. i use SMART clontech kit to do 3' and 5' RACE PCR . i tried so many times, but i got no results, sometimes i got many bands and usually smear. i worked on total and messenger RNAs. i changed everything in the pcr conditions even i used touchdown and nested pcr.
i think my primers is ok. they are 28nts, tm 73 and they give one distinct band when used together. my RNA concentration is to low (2-5ug). my expected 5' is 1kb and the 3' 4kb.
i don't know what to do. is there is a problem in my samples. or should i change the kit.
can anyone help me, please
can you give a bit more details for your PCR?
The SMART clontech kit is ok to use I would say. I used it also before.
Are you sure that your first strand cDNA synthesis is ok?
For the RACE PCR I would generally recommend to try the nested PCR technique as this should make it much more specific.
I suppose that the smear appears in the first (and maybe only round?) round of amplification. Might be that you use too many cycles.
It's hard to say without more details; but what I did was to perform a nested PCR for the 3'-RACE:
Let's say you start with around 20 cycles in the first round and go for 25-30 cycles in the second round of the nested PCR. It might still be that you get more bands in the second round of the nested PCR but then you can still play with specificity of the products.
thank you vey much for your kind reply.
how to be sure of the first strand cDNA? i tried it by my primers and got the target band.
i then made pcr using clontech2 advantage kit. i start with inital denaturation for 2 minutes and then 94 fo 30sec, 66 for 30sec and 72for 3 minutes for 30 cycles, then final extesion for 7 minutes.
i did nested pcr by taking 1-5ul of this product (without purification) and do the same for 25 cycles. i got smear also.
i used the forward primers for the 3' and the reverse one for the 5' (10uM).
sometimes i did touchdown (the protocol of the kit).
i hope you can help me
Well, I can just tell you what I did and maybe this is also working for you though the primers and products are different:
I synthesized cDNA accrding to the RACE SAMRT cDNA amplification kit from clontech, diluted this cDNA 1:11 in TE-buffer and used 2,5µl for the first round of amplification (i think it were 20-25 cycles). Check by agarose gel if you can detect anything (it's even better if you do not...).
Afterwards I diluted the first PCR products 1:50 in TE buffer and reamplified with another 25 cycles using the appropriate nested primers. According to the temperatures a.s.o you have to play a little but I assume that your approach is not working specifically because you use too much of product in the beginning and result in smear in the end. I also had smear in the beginning and mainly changed the amount I used for the respective PCRs.
Once you have distinct bands in your samples you can go and adjust temperature etc... .
thank you very much. ONE more question please; did u work with mRNA or t-RNA? because smearing increased on using mRNA, so, i prefer to use t-RNA. Is it ok?
My primers were designed for mRNA or cDNA respectively