single stranded binding protein? - how to put into my PCR reaction? (Dec/24/2008 )
I am thinking of using single stranded binding protein (SSB) because I've heard it can improve PCR reactions. Has anyone here used it in their PCR? I am wondering how to actually use it? I am not sure how to add in the protein without changing my standard 5ul reaction volume.
I will dilute the SSB stock to my desired concentration with the buffer they recommend which is a mix of Tris-HCl, NaCl, and DTT. I am thinking of starting with 100ng of the protein into my 5ul PCR reaction.
Do I just replace the water step in my PCR reaction with 100ng SSB? That is, if I usually add in 2ul of water into my PCR, can I just replace that with 2ul of 100ng SSB in buffer?
Umm.... yes.... but...
Given that your PCR volume is so small, have you looked up how much does the SSB buffer differs from the PCR buffer? pH? Ion concentration. Will the SSB be happy, and will the DNA polymerase be happy. The component most worrying is the NaCl, what is the concentration in the SSB buffer and the PCR buffer. You might want to increase the DTT, as it would be dilute upon addition to the PCR buffer.
I am curious, which species of archea did you get the SSB from?
How big is your PCR product that you are aiming for? Something big I guess.
Best of luck!!! Tell us how it goes. Send us pictures.
Given that your PCR volume is so small, have you looked up how much does the SSB buffer differs from the PCR buffer? pH? Ion concentration. Will the SSB be happy, and will the DNA polymerase be happy. The component most worrying is the NaCl, what is the concentration in the SSB buffer and the PCR buffer. You might want to increase the DTT, as it would be dilute upon addition to the PCR buffer.
I am curious, which species of archea did you get the SSB from?
How big is your PCR product that you are aiming for? Something big I guess.
Best of luck!!! Tell us how it goes. Send us pictures.
hello,
My regular PCR buffer consists of KCl and Tris-HCl and is optimal for the DNA polymerase I use so that the buffer and polymerase are very happy together. The NaCl is in mM concentration as is the KCl in the PCR buffer. I am wondering if I can just dilute the protein in water? I am using SSB from E. coli strain, called ET SSB. Not sure what archea, just that it is very thermophillic.
Can I just dilute the ssb in water and forget about the buffer they recommend? The Tris-HCl, NaCl, and DTT mix is listed as a storage buffer, not as a dilution buffer. Any thoughts?
don't dilute your protein in water. It can cause the protein to denature.
Ok.
So if my PCR buffer and the SSB buffer both have a salt (KCl, NaCl), should I be worrying about anything?
The regular PCR buffer I always use has 500mM KCl and 150mM Tris-HCl.
The SSB storage buffer they have has 20mM Tris-HCl, 200mM NaCl, 0.5mM DTT, and 1mM EDTA, pH 7.5. What kinds of effects does DTT and EDTA have on PCR reactions?
Again, I was thinking that I can use this storage buffer as the dilution buffer. Another idea is to use regular PCR buffer as the dilution buffer for SSB. That way, I know the buffer will be 100% compatible with my DNA polymerase. Not sure how well it will work with the SSB. Any idea?
I can't find any protocols on this!
DTT has no effect to a positive effect on PCR. Keep it
EDTA is bad for PCR as it chelate Mg2+ ions. Best to leave that out.
Na / K...I am uncertain of the effects of K+ and Na+ ions.
The PCR buffer formulation quoted looks like a 10x concentration mix. It is too concentrated.
Is the SSB buffer at 1x concentration really 200mM NaCl and not 20 NaCl?
I guess if you have a the reagents, one way to find out is to do an experiment. Dilute a small amount of your SSB protein in PCR buffer and run a test PCR with and without SSB protein.
EDTA is bad for PCR as it chelate Mg2+ ions. Best to leave that out.
Na / K...I am uncertain of the effects of K+ and Na+ ions.
The PCR buffer formulation quoted looks like a 10x concentration mix. It is too concentrated.
Is the SSB buffer at 1x concentration really 200mM NaCl and not 20 NaCl?
I guess if you have a the reagents, one way to find out is to do an experiment. Dilute a small amount of your SSB protein in PCR buffer and run a test PCR with and without SSB protein.
The PCR buffer is 10x yes. The SSB buffer is not listed as 1x or 10x concentrations. It just gives the reagents and their concentrations. NaCl is 200mM. Tris-HCl is the one that is 20mM.
I guess I can make a PCR and test with SSB in PCR buffer and without as well as their SSB buffer. I can stick some control DNA in there too to perform a normal PCR and look for any bands on a gel.