2 replies to this topic

[manraj]

Hi all,

I am facing a problem with annealing my complementary oligos (60-mer each). I have tried using different annealing buffers without any success;
1. 100mM NaCl + 50mM HEPES pH7.4 Or
2. Made up 10X 100mM Tris HCl pH7.5, 1M Nacl and 10mM EDTA and diluting down to1X in MilliQ water).

What I am trying to do is to insert my annealed oligos into a pSUPERIOR.neo  vector system for inducible expression of siRNA.

I add equal equimolar or both oligos in annealing buffer (50µl final volume) and incubated the mixture at 95OC for 5 min in a PCR machine and -1OC/min till 4OC.

The sequence of my oligos are as follow;

5’GATCCCCCCGCAACAACCAGGGCACTTTCAAGAGAAGTGCCCTGGTTGTTGCGGTTTTTA3’ and

5’GGGGGCGTTGTTGGTCCCGTGAAAGTTCTCTTCACGGGACCAAGAACGCCAAAAATTCGA3’

Can anyone please help me? Can there be an issue with the Tm of each oligos? Any suggestions?

Many thanks and your help is truly appreciated.

Regards,
Raj

[stardust]

Hi there,

I think your Oligos ar wrong...in the double strand it should be

5`-------------3`
3`-------------5'

Your Oligos bind

5'--------------3`
5'--------------3'

In my opinion they can't anneal ever...

Stardust

The sequence of my oligos are as follow;

5’GATCCCCCCGCAACAACCAGGGCACTTTCAAGAGAAGTGCCCTGGTTGTTGCGGTTTTTA3’ and

5’GGGGGCGTTGTTGGTCCCGTGAAAGTTCTCTTCACGGGACCAAGAACGCCAAAAATTCGA3’

[rkay447]

These will never anneal.  You need to have one primer as a reverse compliment.  What you have here is simply a compliment strand but not reverse.  DNA anneals with a 5' on one strand to the 3' end of the other.  I don't think this is the best explaination but think of all the text models:

5' DNADNADNADNADNA 3'
3' ANDANDANDANDAND 5'  

What you are trying to create with this set of primers is:

5' DNADNADNADNADNA 3'
5' DNADNADNADNADNA 3'

You are also clearly trying to add overhangs for ligation.  Make certain you get the correct sequence on the reverse compliment or it will never ligate, even if you get good annealing.  For example, BAMH1 is GGATCC and the enzyme cuts between the two G.  Now your vector DNA before looks like:

GGATCC
CCATGG

After digestion it is:

5' ---GATCC 3'
3' ----------G 5'

The reverse compliment primer needs to start with GATC to fill in the overhang in a 5'-3' manner.