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Mutagenesis


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#1 moerae

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Posted 22 July 2010 - 07:32 PM

Hi,

I've never done site-directed mutagenesis before and have purchased pfu turbo taq pol (Stratagene) and DpnI and primers. I'm trying to follow the QuikChange Site-Directed Mutagenesis manual and have come to a bit of a stopping point. This might sound stupid, but how much dNTP do I use? The kit comes with a dNTP mix which is "proprietary", does anyone have suggestions? And how much DNA template should I start with? I'm using a plasmid.

Thanks in advance.

#2 phage434

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Posted 22 July 2010 - 09:12 PM

Rumor has it that the "proprietary mix" is simply a higher concentration, perhaps 2x. No one knows, I don't think.

#3 bob1

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Posted 25 July 2010 - 02:48 PM

I just used the standard PCR mix concentrations, it should work fine.

#4 moerae

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Posted 25 July 2010 - 07:25 PM

I just used the standard PCR mix concentrations, it should work fine.


Okay. I'll try that. How about the amount of DNA to add? The protocol says to try different concentrations of DNA ranging from 5 to 50 ng.

#5 moerae

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Posted 26 July 2010 - 02:54 PM

Sorry.... have another question regarding the protocol, this time to do with the DpnI digestion. The manual says to just add 1 uL of the enzyme straight into the reaction mixture but doesn't mention the addition of the reaction buffer that goes with DpnI. Does that mean I do not need to add it?

#6 bob1

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Posted 26 July 2010 - 03:45 PM

A range of DNA concentrations is to make sure that the PCR is working. I think I used 10 ng, but this would vary depending on the size of the plasmid+insert you are mutating.

Dpn1 works well in pretty much any standard buffer, including PCR buffer, you do not need to add any other buffer or clean up your PCR before Dpn1 treatment.

You may find that a ligation step before the Dpn1 step will increase your chances of success, in which case you should clean up the PCR, ligate, then Dpn1 digest.

#7 moerae

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Posted 26 July 2010 - 06:12 PM

A range of DNA concentrations is to make sure that the PCR is working. I think I used 10 ng, but this would vary depending on the size of the plasmid+insert you are mutating.

Dpn1 works well in pretty much any standard buffer, including PCR buffer, you do not need to add any other buffer or clean up your PCR before Dpn1 treatment.

You may find that a ligation step before the Dpn1 step will increase your chances of success, in which case you should clean up the PCR, ligate, then Dpn1 digest.


Hi thanks for the answers. Sorry to keep asking questions, but when you said adding a ligation step what do you mean?

#8 bob1

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Posted 27 July 2010 - 04:09 PM

Basically the Quikchange system is not an amplification reaction, it is just replicating the parent plasmid with some error-incorporating primers, and as the polymerase is not a ligase, the PCR goes right around the plasmid, but can't displace the primer and strand that is already there, so the ploymerase falls off the plasmid, leaving a nicked circular plasmid. If you then ligate the nick so that the plsmid is closed, it will be more efficient for replication after the transformation.

#9 moerae

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Posted 28 July 2010 - 06:17 PM

Thanks for telling me that. Is the ligation step just a standard ligation reaction? How much of the cleaned up PCR mixture should I use for the ligation step?

#10 bob1

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Posted 29 July 2010 - 02:00 PM

Yes, the ligation is a standard ligation. I would use as much of the PCR as you can, unless you want to keep some as a control or to use for digestion as per the standard proceedure.




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