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