I would like to prepared a DNA sample that has -SH or thiol group at one of its end so that I can covalently immobilize it on a surface. The -SH modified primers are commercially available.
I am purchasing a 5'-SH modified forward primer and unmodified reverse primer and have the template. I have Phusion High fidelity enzyme for PCR. But I don't know if regular PCR protocol would work for this purpose. Please provide me the relevant information.
Primer with 5'-SH modification does not cause any problem for PCR. It will work the same as the non-modified primer does.
Hi Ziguo Zhang,
Thanks for your reply. I would like to clarify more details.
I am using Phusion High Fidelity Polymerase from New England Biolabs (NEB). I am using the PCR protocol suggested by them. For a 50ul reaction mix, the composition is 29ul Sigma water, 1X Phusion HF buffer, 0.2mM dNTP, 3.5mM MgCl2, 0.25uM forward primer, 0.25uM reverse primer, 30ng template (2.3kb), 0.5ul Phusion polymerase.
1. I would like to know if any additional components are required for the PCR mix with 5'thiol-modified forward primer.
2. Would the -SH group be stable after the purification?
3. And can we purify the PCR product which is thiolated dsDNA using Qiagen kit?
Sumanth,
You can do it. But in case if you need to modify the protocol, you van refer this literature: http://pubs.rsc.org/en/content/articlelanding/2014/ra/c4ra04671d#!divAbstract
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In principle there is no problem. Regarding your detailed protocol, there are plenty of modifications you can do to have success. Q5 or Herculase are also good polymerases. Dependent on template and size, they work sometimes better. You can also modify the MgCl2 concentration or test different annealing temperatures. In case I'm in a rush for some cloning, I use different polymerases with there respective buffers, put 3 rows on the gradient cycler and normally have a positive result within a day.
Yes u can
As the thiol gp (SH) has the same chemical characteristics of 3OH gp on the primer; do the nucleotides are easily added to the primer to synthesize the DNA strand.
As a general rule, 5' modification of the primer has no effect on PCR.
3' modification, on the other hand, is usually disastrous.
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