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multi-site mutagenesis


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#1 Opal

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Posted 15 May 2011 - 10:04 AM

I'm going to mutate multiple sites of the gene which I have already cloned into a 4.5 kb plasmid (plasmid+gene).My search results show that the "QuikChange Multi-Site Directed Mutagenesis Kit" from Stratagene is probably the best way to go! I'm wondering if there is any way so that I don't have to buy the whole kit and just purchase the pfu Ultra (or an enzyme with similar features). I know that I can buy pfu ultra separately, but is it exactly the same thing provided in the kit as the enzyme blend with a nick sealing capacity? please share your experiences with me !!!

#2 GNANA

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Posted 15 May 2011 - 01:22 PM

When i did, i didnt buy the kit, just did with Accuprime pfx polymerase and dpnI enzyme...you can very well manage without buying the whole kit.

Gnana...
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#3 Opal

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Posted 15 May 2011 - 06:16 PM

When i did, i didnt buy the kit, just did with Accuprime pfx polymerase and dpnI enzyme...you can very well manage without buying the whole kit.

Gnana...



May I ask for the protocol you used for the multi-site mutagenesis? The protocol provided by Strategene requires simultaneous polymerization and ligation of the DNA which can not be done by a DNA polymerase alone!

#4 Rsm

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Posted 15 May 2011 - 10:37 PM

You have to have 5' phosphorylated primers and a heat-stable ligase. Protocol is very similar to Stratagene kit, you can use it as starting point.
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#5 GNANA

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Posted 16 May 2011 - 12:24 AM

Taken 50 ng of template and 125ng of each primer and did PCR reaction (14-18 cycles) with Accuprime, then the Pcr reaction was digested with DpnI (37 degree for 1 hr), after Dpn I degestion the entire product was gel extracted and transformed in library efficient competent cells...thats it, sequence it and confirm the mutation,,,,Primers are designed as per strategene suggests...
i have done single or maximum two point mutations (if they are closer) with a single pair of primers, if i got to do multiple mutations that are far apart, i would do sequential Pcr mediated mutagenesis...good luck...

Gnana...,

Edited by GNANA, 16 May 2011 - 12:34 AM.

I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#6 Rsm

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Posted 16 May 2011 - 02:56 AM

If I remember correctly... I used 1ul Pfu polymerase, 100ng template, 0.08ul of 100nM 5'P-Primer each, 4ul dNTP plus 1ul Taq DNA ligase in 50ul total volume. Run at 96C 2min, then 25cycles of 96C 30s - 58C 20sec - 72C 1min/kb, and then 72C for 5min. Add 1ul Dpn1, 37C 1h and run gel to see if you have any product.
I thought it was rather difficult to get a good product. I'd rather recommend overlap extension PCR... you can also do it on multiple loci, and it works more often than SDM.
I got soul, but I'm not a soldier

#7 Opal

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Posted 16 May 2011 - 05:35 AM

If I remember correctly... I used 1ul Pfu polymerase, 100ng template, 0.08ul of 100nM 5'P-Primer each, 4ul dNTP plus 1ul Taq DNA ligase in 50ul total volume. Run at 96C 2min, then 25cycles of 96C 30s - 58C 20sec - 72C 1min/kb, and then 72C for 5min. Add 1ul Dpn1, 37C 1h and run gel to see if you have any product.
I thought it was rather difficult to get a good product. I'd rather recommend overlap extension PCR... you can also do it on multiple loci, and it works more often than SDM.



So you added the pfu and the ligase at the same time, right? I'm just going to do two mutations; they aren't close enough to be embedded in the same primer; however since they are in tandem repeats I guess if I design one single primer and add enough of it to the reaction it is going to target both sits! this is my hope ...

#8 Rsm

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Posted 16 May 2011 - 11:28 PM

Yes, I added the ligase at the same time. You'll need a heat-stable ligase (and 5' phosphorylated primers), otherwise it won't work. Tandem repeats are tricky stuff, good luck to you.
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