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Hi,
I've been enjoying this site as a reference for tips on trying to get a Quikchange-like point mutation done with primers that were handed to me (and not working). I was wondering if there is a specific reason to heat inactivate the DpnI after the 2hr incubation at 37C? A colleague doesn't bother with that to save time, but I've also come across a statement that it is actually detrimental (see below). I can't really find evidence for that statement so I figured I would ask here why it is included in the RF-Cloning.org protocol.
Link to original: http://laforeta.blogspot.com/2011/10/si … redux.html
"Thermalcycling:
Set up protocols according to the polymerase used.
If there is a final extension step, don't include it.
Do not exceed the extension time specified for your enzyme.
15-18 cycles should be enough, do not exceed 20 cycles.
Following that:
Add excess amount of DpnI, usually 10U per 50uL reaction but feel free to add more if have a lot of template. Not to mention the PCR buffer is usually not optimal for DpnI activity
Vortex and centrifuge the tube to ensure good mixing. Incubate at 37C for 1-2 hours in a PCR machine with heated lid or cover with mineral oil to prevent evaporation. Vortex/spin at least once during incubation.
(Optional) Add Proteinase K and incubate to stop the reaction. __Do not heat inactivate since this might interfere with strand pairing.__"
Overall, I think my Quikchange reaction problem (no colonies) is shorter than ideal (22nt) completely overlapping primers with a mismatched codon in the center. I've heard both sides that Phusion is compatible with completely overlapping primers and is a poor choice for said primers. Since Phusion is all I've got, I'm currently going to 3%DMSO and/or GC Buffer with broad temperature scanning via gradients to try to get something to work before re-ordering primers. I just wanted to check that the DpnI heat inactivation step I was not causing me any problems unnecessarily.
Thanks for your help! Great website!
Best,
ChrisC
Last edited by ChrisC (2017-08-28 07:49:51)
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Hi Chris,
I don't remember explicitly testing with and without heat inactivation. That said, I don't intuitively understand why strand pairing would be affected; heat inactivation occurs well below melting temp of the ds-plasmid, so any mispairing will already be an issue following the final PCR cycle. From my own experience, small inserts and point mutations almost always worked with the vanilla RF-Cloning protocol outlined in the manuscript as long as there wasn't some confounding issue in the plasmid (e.g., it was very large, contained a tandem repeat, etc.).
Do you have a positive control that is working for you?
Best,
-Steve
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