PCR with Betaine, DMSO, BSA, DTT - (May/08/2008 )
Attached is a gel with a series dilution, ever other sample has an enhancer cocktail added to the PCR reaction (1-100%, 2-100% w/ enhancer, 3-20%, 4-20% w/ enhancer,...)
As you can see, the enhancer is having a negative effect? The weird thing is the enhancer initially was showing great promise, it was really enhancing products! Can the enhancer go bad? I have stored it at -20 (it doesn't however freeze solid at that temperature). I don't remember reading anything about making it fresh every time or any specific storage conditions? Any ideas? One difference is I have increased my denature temp(from 94 to 99) in the PCR reaction, could that reverse the enhancers effects? Also if you look at the gel, each 'enhancer lane' shows flourescence around the loading wells?
I got the enhancer recipe off some post here.
2.7M Betaine 540μL 5M Betaine
6.7mM DTT 6.7μL 1M DTT
6.7% DMSO 67μL DMSO
55μg/mL BSA 55μL - 1 μg/μL BSA
DTT is probably the main chemical that will go off there as it becomes oxidised. But in all honestly, the reason for the enhancer inhibiting your PCR is that one of the reagents in the enhancer is inhibiting the PCR. The effective ingredients in your enhancer there are betaine and DMSO; DTT and BSA are essentially doing nothing in terms of getting your product. The important thing to remember is that betaine and DMSO are only useful if your template is GC-rich (~55% or greater). Both help to dissolve secondary structure that prevents the polymerase from reading through those regions. If your template is less than 50% GC, these reagents will inhibit your PCR. I suspect DMSO is causing your drop off in signal because it works by interfering with hydrogen bond formation, which dissolves your secondary structure but also interferes with primers annealing to the template (i.e. drop off in signal). Betaine is an isostabilising agent and works by equalising the strength of GC and AT bonds so that GC bonds are not as strong or more specifically AT bonds are stronger. Betaine is a much better reagent that DMSO. I think you'll find betaine by itself works the best. Another thing to add if you want to improve yield (not specificity) is an extra 4 mM MgCl2. Magnesium chloride strengthens all hydrogen bonding because it is chelated by nucleotides, so this strengthens primer annealing to your template. It will also strengthen secondary annealing but if all you want is your PCR product then that is fine and your bands are far enough apart there. So if your GC% is high, use a gradient with DMSO (0 - 10%) and betaine (0 to 2M) individually and add the 4 mM MgCl2 (if you want to save time just do the betaine + 4 mM MgCl2). If your GC% is low/lowish, forget the betaine and DMSO and just add the 4 mM MgCl2.
Good luck, Rob
Thank you for an excellent response!
I tried making fresh enhancer and compared it with the 'old' enhancer and with no enhancer. The 'old' and fresh were the same, so I don't think it was bad.
Your explanation confirmed what I thought may have been happening. The region is very GC rich, that is why I am using the enhancers. I guess they help when my denature temp is low, but once I raised the temp, then all the enhancers weren't helping. I guess Mg may be the way to go.
In that case, follow my GC-rich advice (betaine gradient + 4 mM MgCl2) and you should see better success.
Hi, do you mean to add an additional 4mM? on top of the 2mM MgCl2 in the PCR Buffer? or just add 2mM so the total concentration of MgCl2 is 4mM?
Sorry, i should have said up to 4 mM MgCl2 (an additional 2.5 mM MgCl2), most PCR buffers have a final concentration of 1.5 mM. So add 2.5 uL of 50 mM MgCl2 to a 50 uL reaction. Never added an additional 4 mM MgCl2 before but i wouldnt be surprised if it worked well too.