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Quikchange Mutagenesis Help Please!!!


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#1 carvixki4

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Posted 11 June 2009 - 09:59 AM

Hello everyone,
I am attempting to create a single base mutation using the standard Quikchange mutagenesis protocol followed by DpnI digest for an hour at 37 degrees C, then transformation into TOP10 cells.
Anyhow I've been trying this for several weeks now, with no results, meaning that I am not getting any colonies after transformation --- the problem is not the transformation itself since I have run controls, but it is in the PCR reaction. Anyway my Tms of my primers are close to 78 degrees and are about 30 base pairs in length.
I have ran a gradient (50 - 70) of annealing temperature, I have changed DNA plasmid concentration that I have used (lowered/raised) and pretty much changed every possible variable. Anyway if anyone has experience with this I really could use some help!!
Thanks so much,
Vinny

#2 Michaelro

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Posted 11 June 2009 - 11:23 AM

Hello everyone,
I am attempting to create a single base mutation using the standard Quikchange mutagenesis protocol followed by DpnI digest for an hour at 37 degrees C, then transformation into TOP10 cells.
Anyhow I've been trying this for several weeks now, with no results, meaning that I am not getting any colonies after transformation --- the problem is not the transformation itself since I have run controls, but it is in the PCR reaction. Anyway my Tms of my primers are close to 78 degrees and are about 30 base pairs in length.
I have ran a gradient (50 - 70) of annealing temperature, I have changed DNA plasmid concentration that I have used (lowered/raised) and pretty much changed every possible variable. Anyway if anyone has experience with this I really could use some help!!
Thanks so much,
Vinny

Hi
It looks to me that You may have problem with the template
Try to sequence your template plasmid to make sure that You use the right thing.
Since the Quick change mutagenesis generally works very well.
Also I'd try different enzyme stock to make sure that the polymerase works.
Good Luck

#3 carvixki4

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Posted 11 June 2009 - 11:49 AM

Hi Thanks so much for your reply,
I previously thought of that and sequenced the template - and everything looks fine. We ordered new pfu turbo polymerase enzymes and new dntps etc..and still no good results. I've redesigned my primers three times now and, honestly, I'm kind of confused what to do at this point.

#4 mastermi

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Posted 11 June 2009 - 12:00 PM

Maybe there is something wrong with your DpnI.
I never heard of having no colonies after QuikChange, because usually the DpnI digestion doesn't work perfectly, so that you will still have little template left.

I once needed 4 primer pairs until the QuikChange finally worked, but I always had some colonies after transformation, just that they were template and not changed...

#5 phage434

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Posted 11 June 2009 - 04:53 PM

You could make additional primers that will amplify a small fragment when paired with one or the other of your mutation primers. This would show that your mutation primers are correctly binding.

Make sure you are not using a strand-displacing PCR enzyme.

Test the competence of your cells. Being able to transform high concentration plasmid is very different from being able to clone with a competent cell prep. You should get a lot of transformants with 10 pg of plasmid DNA.

#6 Michaelro

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Posted 12 June 2009 - 02:41 AM

Hi Thanks so much for your reply,
I previously thought of that and sequenced the template - and everything looks fine. We ordered new pfu turbo polymerase enzymes and new dntps etc..and still no good results. I've redesigned my primers three times now and, honestly, I'm kind of confused what to do at this point.

I'd try to perform mutagenesis reaction on diferent plasmid that works in order to see that the reaction itself works fine

#7 phage434

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Posted 12 June 2009 - 06:55 AM

Another possible problem would be failure of the PCR reaction due to very high or very low GC content. For high GC, add 5% betaine to the PCR reaction. For low GC content, lower the extension temperature to 62-65 C.

You could check the length of the PCR fragments on a gel.

#8 carvixki4

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Posted 30 June 2009 - 09:31 AM

Hey everyone, I ordered HPLC purified primers and extended my elongation time to 2.5min/kb plasmid and my quikchange worked. Thanks for all your help.




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