Side directed mutagenesis with 17kb plasmid-Molecular Bi

Hey everybody,

I am working on a side-directed mutagenesis with the Stratagene℃ quickchange mutagenesis xl-kit. Here is my problem: My plasmid is 17kb big. Until now I have had no colonies on my plate. According to Stratagene℃ protocol, I used the following cycling parameters:

1. 95℃ 1 min

2. 95℃ 50 sec

3. 60℃ 50 sec

4. 68℃ 1min/kb plasmid (so for my plasmid 17min)

5. 68℃ 7min

As I have already said no colonies! Then I read that the Pfu-polymerase needs 2min/kb of the plasmid in the elongation time. So I increased the elongation time from 17 min to 34 min. But again no success. I would like to ask if anyone has got an idea.

Thanks in advance!!!

-toutzari-

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This is going to be difficult! You might have a high G+C region that the polymerase can't get through. Another apporach might be to try and mutate the plasmid in vivo using a genomic site directed mutagensis approach. Have a look at this paper

-RWhity-

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Can't you cut out the region you want to change and clone it into a smaller vector (like pUC, pBluescript,...), perform your mutagenesis and clone it back into the original one?

-vairus-

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Transformation efficiencies are going to be a problem. You will need to electroporate or subclone to get it below 10 kb.

-Matt

-MisticMatt-

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Hi!

Thank you all for you help! I think the transformation effiecency is not the problem, I already transformed such big plasmids. I think there is a problem with the polymerase, I cant hardly believe that an elongation time of 34 min will work out... I checked it for digestion and cloning in a smaller part but its looks not so good. Do you think more cycles (e.g. 22-25) would help my situation?

Thank you all again!!

-toutzari-

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You can try the 'improved' version of Pfu and PfuTurbo (which already is an improved version of Pfu), named Pfu Ultra. I've gone up to 9 kb in SDM with this one and it worked out...

-vairus-

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QUOTE(toutzari @ Dec 12 2005, 01:09 AM) [snapback]34193[/snapback]

Hey everybody,

I am working on a side-directed mutagenesis with the Stratagene℃ quickchange mutagenesis xl-kit. Here is my problem: My plasmid is 17kb big. Until now I have had no colonies on my plate. According to Stratagene℃ protocol, I used the following cycling parameters:

1. 95℃ 1 min

2. 95℃ 50 sec

3. 60℃ 50 sec

4. 68℃ 1min/kb plasmid (so for my plasmid 17min)

5. 68℃ 7min

As I have already said no colonies! Then I read that the Pfu-polymerase needs 2min/kb of the plasmid in the elongation time. So I increased the elongation time from 17 min to 34 min. But again no success. I would like to ask if anyone has got an idea.

Thanks in advance!!!

hello,

did you check on a gel if you amlified something?

-laurence-

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Hey,

no I didn℃ check it on a gel, I think it is not possible to see the difference between my template and the amplification product (they should have the same size) on the gel . Now I tried a mutagenesis (same kit and protocol) with a 7kb plasmid and again no success... I think I am doing the same mistake the whole time...

-toutzari-

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You can check on gel, do your PCR (35 cycles) and put it on a gel (say 5 ℃ of a 50 ℃ reaction), and right next to it, put your template (and make sure you also dilute your template to a 50 ℃ total volume and put 5 ℃ of it on a gel). You should then easily see a difference.

Also, transform your PCR-reaction directly, without the cuttin part... Check how much colonies you get and compare it to a similar dilution of your template...

-vairus-

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QUOTE(vairus @ Dec 15 2005, 06:59 AM) [snapback]34624[/snapback]

You can check on gel, do your PCR (35 cycles) and put it on a gel (say 5 ℃ of a 50 ℃ reaction), and right next to it, put your template (and make sure you also dilute your template to a 50 ℃ total volume and put 5 ℃ of it on a gel). You should then easily see a difference.

Also, transform your PCR-reaction directly, without the cuttin part... Check how much colonies you get and compare it to a similar dilution of your template...

You can try a mix of PFU and TAQ too. I've read that it greatly improves the elongation. I don℃ know the exact quantities though.

Best regards,

-carlatf-

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This will decrease your accuracy. And with site directed mutagenesis you want to have the smallest amount of extra mutations as possible. Also, I'm not sure if TAQ has strand displacement activity, which pfu doesn't have @ 68℃ (not having strand displacement is necessary for mutagenesis).

-vairus-

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You might want to try the Phusion enzyme from Finnzyme/NEB. It has high processivity and good performance on long templates.

-phage434-

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