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Dpn1 mutagenesis


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6 replies to this topic

#1 ariaz

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Posted 26 October 2010 - 06:29 AM

Hi every one,
I am very new to mutagenesis.I am trying to delete 8bps from my plasmid, but couldnt get any colonies.
I have a few things (rather a lot of)which i am not sure about. If any one one can help,
1. what would be the effect of increasing or decreasing the anealing temperature to one or two or three degrees.
2. which of the elongation time is good either 1 min/ kb or 2min/kb.
3. When i run the pcr product on gel there was a faint band but when digested it with dpn1 and then run it again, there was nothing. any idea?
4. I am using one shot top10 competent cells, are these ok for this kind of transformation.
(The quantities of primers and template are according to the stratagene protocol)

Kindly help me.
Thanks

#2 bob1

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Posted 26 October 2010 - 02:44 PM

You could try an annealing temperature gradient, but the results of it may not be obvious as the qickchange method is not really an amplification method as such, it is more about copying the template and creating single strands with the mutation (dpn1 digests the methylated and unmutated parent strand, leaving you with the mutant strand incorporating your primer).

The elongation time is dependent on the polymerase you are using. The manual for the polymerase should be able to tell you optimal times for extension. Remember that you are trying to amplify all the way around the plasmid so if you have a big plasmid, you will need a longer extension time.

Dpn1 digests only methylated DNA, single stranded DNA left over from the digestion does not pick up EtBr very well compared to dsDNA, so it is probably still there, just not visible.

Top10 are perfectly fine for this, as are most competent cells.

You may find that the process is more efficient if you ligate the circular ends of the replicated strand before the digestion step - this will leave you with a circular plasmid that should transfect more efficiently into the bacteria.

#3 ariaz

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Posted 27 October 2010 - 12:16 AM

Thank you very much Bob1. These suggestions will definetely help me.
Before ligation or before transformation is there any need to clean up the pcr product, or this step is optional.
Secondaly Ligation protocol is same as used for other ligations?
Thanks again

#4 phage434

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Posted 27 October 2010 - 05:33 AM

Ligation will not do anything unless the primers used for the PCR have a 5' phosphate. You can either order them that way or kinase them. It's probably not necessary, in any case.

#5 ariaz

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Posted 27 October 2010 - 09:51 AM

Thanks phage434,
I heard that this whole process of mutagenesis, which can be done in one day, should be done in 2 days. And please also tell me, how much PCR clean up is important in this process.
Thanks again.

#6 Curtis

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Posted 28 October 2010 - 12:11 PM

Read this also,

http://www.protocol-...8892#entry88892

#7 ariaz

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Posted 29 October 2010 - 03:08 AM

Read this also,

http://www.protocol-...8892#entry88892

Thanks. Its a very useful information in there.
I did 2 mutagenesis experiment for my template using same stratagene protocol for one and for other i did a few changes, like i increased elongation time from 6 min to 10 min , and i also did pcr purificcation. I got colonies in both cases but 100s in first and a few in 2nd.
Another thing, when i run samples on gel before Dpn1 digestion and after Dpn1 digestion i got bands only for first experiment and no bands for second one(neither before nor after dpn1 digestion)..Any ideas about all of this ??????? :huh:
I still have to miniprep it and i am hoping i will get required result.
Thanks in advance




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