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Problem with Site-directed mutagenesis of a 9 kb template with high GC content (

molecular biology site-directed mutagenesi

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#1 MT78

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Posted 08 April 2014 - 01:03 AM

I've been trying to mutate a single base in a long template (9kb) with high GC content with the QuickChange kit without good results. I followed the protocol exactly as Stratagene says since I've done it before with smaller templates and I always got good results. I've designed the primers with the manufacturer tool and optimized them since the ones they suggested could form hairpins. I know that the problem is in the PCR since I get results with the control reaction of the kit but nothing with my sample. I've seen this on a gel after the PCR and I'm aware that a band may or may not appear, so I continued with transformation and no results there either with my sample but many colonies with teh control. I am aware that I'm pushing the kit, since it's not the XL but the regular one, concerning the size of the template. I've used also 10% DMSO to counteract the GC difficulties and no results either. Does anybody know if it is possible to use this kit for 9 kb templates and high GC content? The last thing I can try is a 2 min extension/kb (18 min for my template) but I don't know if that will work either. I'll be very happy to read your suggestions or questions if I forgot to mention something...



#2 Trof

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Posted 08 April 2014 - 03:14 AM

Did you used only 10% DMSO for this or lower concentrations too? (in my experience, 10% DMSO inhibits PCR quite a lot, got much better results generaly with 5 % DMSO)

 

If you didn't, try 1–10%  range of DMSO (like 1, 3, 5, 7, 10 for example) or try another additive, betain in 0.5–2.5 M range (Sigma sells 5M PCR solutions). 

If the problem is only GC content, this should work.

 

When you're not sure if your PCR is working, use more cycles than in the protocol (common 30x instead of 10-15x in QuickChange protocol), this way you should be able to see the product on gel first (may require actually more than 30 cycles with 9 kb, not sure). If you see it, you can then compare the efficiency between different amounts of additives (the number of cycles should not be too high, then you may not see difference, you can play with this though, since this is just PCR, costs no cells).

 

After you find the best PCR conditions, you do it as in the QuickChange protocol (lower number of cycles only), digest etc. and transform. Don't waste your competent cells unless you're sure you have PCR product.

Good luck!


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I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#3 MT78

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Posted 08 April 2014 - 04:25 AM

Thanks for your input and help! smile.png

 

Indeed I haven't tried other DMSO concentration, so I think it's definiteley worth the try with different concentrations.

I will also increase the number of cycles as your are the second person suggesting this today wink.png

 

Hoping to finally get a band in a gel!!!

Maria



#4 Trof

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Posted 08 April 2014 - 06:11 AM

Yes, but have in mind that reduced cycles in the QuickChange protocol are there because the number of cycles increase a probability of getting unwanted DNA change due to polymerase mistake elsewhere in the plasmid.

 

Since much less DNA is required for successful transformation than for visibility on gel, use more cycles to see your product, but do not use that reaction directly for transformation.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon






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