how can I clone a CYP450? - (Sep/03/2008 )

Good morning, I want to clone a gene expressing a CYP450 protein into pCW. I want to introduce 8 modified aa at 5', a His tag at 3' and the two restriction sites to clone directionally. To do it I've obtained the sequence with two specific primers from total cDNA extract, eluted the product and used as template for a new PCR. In this PCR the FW has a site for NdeI, a region corresponding to 8 modified aminocid and a matching region of 18 nt.
the RW has a matching region of 24 nt, aa region corresponding to 4 His and a site for the HindIII.
this primers are too long, they have a matching region of about 20 nt and a flanking region of about 30 nt. when I do the PCR (at differnt Tm and at different amount of template) I can't observe my expected product.
wath is the best strategy to obtain the modified sequence to clone? i know that the primer are too long, but I don't know I can have this sequence in other manner.
Who can help me?
thank u very much for your attention

Fralab

Hi Fralab!
- A flanking region of 30 nt is not long. It is normal to have a flanking region like that and it should not affect your PCR reaction.
- Make sure that you did not loose the PCR product (first reaction) that you are using as a template. Run a gel to check if it is still there/how much you have.
- Also, why not skip the intermediate step and use long primers to amplify CYP450 directly from cDNA?
- Alternatively, if you get really stuck, clone the fragment that you already have and then use that clone as a PCR template.
Andriy