5 replies to this topic

[4leafclover]

Hi all

I'm working on site directed mutagenesis and so far have had no luck with PCR at all.

Initially I was using the stratagene kit, ( it involves PCR with Pfu followed by Dpn digestion of template and transformation), but got almost no amplification bands, the bands that appeared looked like they corresponded to the template that I added. Anyhow, upon transformation, I did get colonies but they all turned out to be WT & not mutant.

I'm trying out Taq now but am just getting entire smear bands. I see faint bands at the end of my lanes, I'm told they correspond to primer dimers, how do you get rid of these, I'm introducing a single nt change in the sequence, the rest is all WT, so how can I get rid of dimers?

I'd appreciate any advise.

I've attached a pic of my gel using Pfu. Lane3: template, 4,5&6: PCR pdts.

Attached Thumbnails

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[beenyg]

Hi,

I've used stratagene's protocol several times, with pretty good success. The truth is, I never even check to see if I have an amplified band, since 16-18 cycles is often to little to see anything in gel.
1) Make sure you are giving enough elongation time.
2) I was having alot of trouble, and found out my competent bugs were not so competent.
3) Primer dimers are an inherent problem with this method. One way that you may be able to improve your results is by first preparing 2 half reactions, each containing all rxn components, but only one primer. Run 1-3 cycles, and then mix the two halves and run another 16 cycles. This allows the primers to create perfectly matched templates, and may help. (This is a published method, I don't remember who published it).

Hope this helps,
Benny.

[phage434]

Taq and Taq containing blends will  never work with the Stratagene protocol.  Taq displaces the 5' end of strands it encounters, producing concatamers from circular DNA.  Pfu and other similar enzymes stop when encountering 5' bound DNA, an important feature for the success  of Quikchange mutagenesis.  Don't run gels, they will just confuse you.  Have you used sufficient template?  You need much more than a normal PCR if you amplify only 18 cycles.  Might I suggest reading the manual?

[4leafclover]

Hi

Thank you very much for your replies.

I have another question:
Why does the stratagene method require complementary primers. When I was trying to figure out what went wrong (I still am) someone told me that primers are NEVER designed complementary. Has anyone ever designed non-complementary primers with this method?

Could primer dimers severely affect my reaction or can you get product even though you have dimers.

I'm thinking of redesigning my primers, non complementary this time. Any suggestions if this is a good idea?

[4leafclover]

phage434, on May 26 2010, 10:44 AM, said:

Taq and Taq containing blends will  never work with the Stratagene protocol.  Taq displaces the 5' end of strands it encounters, producing concatamers from circular DNA.  Pfu and other similar enzymes stop when encountering 5' bound DNA, an important feature for the success  of Quikchange mutagenesis.  Don't run gels, they will just confuse you.  Have you used sufficient template?  You need much more than a normal PCR if you amplify only 18 cycles.  Might I suggest reading the manual?

Hi phage

I have read the manual. I'm using a high template concentration of approx 100ng, with the recommended 125ng primer conc. I was wondering whether to decrease the template conc 'coz someone told me that if I see my template on a gel the conc is too high..Maybe I won't run a get the next time..like u said!

Thanks for the info about taq that probably explains the smear bands...

I'v put a post below about primer designing..let me know what u think..

[4leafclover]

beenyg, on May 26 2010, 04:25 AM, said:

Hi,

I've used stratagene's protocol several times, with pretty good success. The truth is, I never even check to see if I have an amplified band, since 16-18 cycles is often to little to see anything in gel.
1) Make sure you are giving enough elongation time.
2) I was having alot of trouble, and found out my competent bugs were not so competent.
3) Primer dimers are an inherent problem with this method. One way that you may be able to improve your results is by first preparing 2 half reactions, each containing all rxn components, but only one primer. Run 1-3 cycles, and then mix the two halves and run another 16 cycles. This allows the primers to create perfectly matched templates, and may help. (This is a published method, I don't remember who published it).

Hope this helps,
Benny.

Hi Benny

I'm allowing extension for 2min/bp as mentioned in the protocol. I've even added a final extension of 12min. I don't know why final extensions are required, the manual does mention it either but I was told its a good idea.

Does anyone know why final extensions are added after all the cycles are complete??