Cloning primer-dimer fragments - (Jan/23/2006 )

Hello!

im planning a weird cloning strategy that im not sure that it will work. i want to insert some loxP sequences flanked by restriction sites in a vector. for that i cut the vector where i wanted to insert the fragment, dephosphorilated it and know im going to clone my insert.

Until here nothing special everybody as already done it...

my insert will be a dsDNA sequence resulting from primer-dimer, this means that i will order one primer reverse and one forward, both phosphorilated and then will in a certing anneling temperature i will make primer dimers that will produce the dsDNA sequence. After that i will pretend to used that as my insert in a 9kb vector.

The rpoblem is that the insert is too small compared to the vector (rectroviral vector) and the fact that i never seen nobody trying to clone as insert a primer dimer sequence.

Does anybody ever did it, or thing that it will work?

Hope to hear from you.

Best regards to you all

Cheers

This sounds similar to cloning ds-oligo into a vector like in shRNA construction. The oligos used are ~50 nt and complementary. After annealing, they form ds-oligo with sticky ends. It is very easy to ligate the ds-oligo into any vector. How long is your primer dimer? Are the primer complimentary?

Want a detailed protocol how to anneal the oligos and how to do the ligation? Check Ambion's shRNA construction manual.

thanks for your reply!

well i will do several clonings..

the primers will go from 12 bases to around 20 bases...they are not fully complementary. this is...there is a complementary region of about 8-12bp but as i want to clone them with stick ends, the ends of the primers are not complementary....

i will check it....

Do you know you can i check the temperature annealing ideal for formation of primer-dimer. It should be around 2-6 C lower then the Tm of the primer with lowest Tm..

Thanks anyway!