Hi,
I annealed two primers at 95C for 10 min and cooled them to room temperature. They look like the figure attached below with a 20mer overlap. I want to fill in the duplexes but as I am using random molecules in the ends I want to avoid biases from PCR and use an extension reaction. What is my best bet?
If you are asking what I think you are, could you use Klenow Fragment or T4 Pol to extend (assuming appropriate 5'-->3' directionality)
See the attached Gibson Assembly method overview. In one step (similar to yours), DNA polymerase is used to extend the 3' end and fill up the gap.
Or, can you just treat your duplex like sticky end restriction site, and try to 'blunt' it using the Klenow Fragment as suggested by Matthew?
Thanks for your suggestions. I am using the klenow fragment as of now. Was throwing out for suggestions. If there is a random sequence, PCR may limit to the sequences it prefers and amplify only those sequences. That could be a bias
Hi ,
This is reagrding your klenow reaction. I have explained earlier the design of my oligos which I anneal at 1mM concentrations at 95C for 10 min and cool down, purify and resuspend in 50ul water. This generally has a concentration of 250 ng/ul. I take about 5ul of this , add 5ul of cut smart buffer, 1.65ul of 1mM dNTP in a 50ul reaction. Leave it at 25C for 15 min on a thermal cycler. Purify this. To check if the extension has worked, I treat it with s1 nuclease which digests ssDNA.
My klenow doesnt seem to work. Is there anything I am missing?
After you treated them with S1 Nuclease, how did you verify them to lead to the results which you thought "your Klenow did not seem to work".
If they filled in duplexes, they become double stranded. S1 nuclease digestion doesn't work in that case. So when it digested my unextended duplexes, I couldn't see them on my gel after nuclease digestion and I was affirmative of it.
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