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    <title>Molecular biology troubleshooting forum</title>
    <link>http://biowww.net/forum/list/5</link>
    <description><![CDATA[Troubleshooting and methods discussion on genomics, gene expression and regulation, genetics topics ]]></description>
    <language>EN</language>
    <pubDate>Sat, 06 May 2006 23:21:51 -0400</pubDate>
    <lastBuildDate>Sat, 06 May 2006 23:21:51 -0400</lastBuildDate>
    <category>Molecular biology troubleshooting forum</category>
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    <item>
      <title>Re: Site directed mutagenesis with PCR</title>
      <link>http://biowww.net/forum/read/5/1065/1922#msg-1922</link>
      <author>besttuti</author>
      <description><![CDATA[For rapid and cost-effective method, I strongly recommend recently published method using type IIs enzyme (Ko, JK and Ma J, Am J Physiol Cell Physiol. 2005, 288: C1273-8.). This method originally published by Tomic M (Nucleic Acid Res. 1990, 18: 1656.).  My colleagues and I also successfully generated various mutants (substitution, insertion, deletion and chimeragenesis) by this method. You need only four primers (just desalted not purified, it¡¯s very cheap!) and type IIs restriction enzyme.  The mutagenesis efficiency is close to 100%.  
Presently, the overlap extension method, megaprimer method, Quick Change method (Stratagene) are prevalent among numerous PCR-based mutagenesis methods developed commercially or non-commercially.  However, all of them has shortcoming for applying to the diverse mutagenesis strategies including base substitution, deletion, insertion, chimeric gene generation and multiple-site mutagenesis.  Especially, they show low mutagenesis efficiency (below 40%) for genes with long sequence (&gt;3 kb), GC-rich region, tight secondary structure, or tandem repeats and for a long frame mutation (&gt; 10 bp). 
If you have plan to use commercial kits, another choice for short way for your research is use of mutagenesis service company.  I realized the mutagenesis cost using commercial kit is never cheap!  My estimate is $200 ~ 250 per one mutagenesis reaction using kit (one kit rexn., $20  + purified two primers, $150 + three clone sequencing, $30 + subcloning reagent, $30 : SM, media, agar plate, buffer, tube, tips --- and time).  Besides, purchasing kit for just one or two mutant generation is money wasting because remaining kit reagents are useless.  Recent biotech companies provide rapid and precise mutagenesis service in affordable prices.  Some people in our lab use Mutagenex Inc. (USA) and I found other companies offering in low price:
Mutagenex: $249 per mutation, USA
Topgenetech: $269 per mutation, Canada
MCLab: $280, USA 
You can also find more other companies that have different technology and service criteria.
In conclusion, my recommendation is,
		Efficient and cheap method: Type IIs method! 
		Easy way but need money: company rather than kit!
For more discussion, you can contact to choik1@umdnj.edu
]]></description>
      <category>Molecular biology troubleshooting forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/5/1065/1922#msg-1922</guid>
      <pubDate>Sat, 06 May 2006 23:21:51 -0400</pubDate>
    </item>
    <item>
      <title>Re: Site directed mutagenesis with PCR</title>
      <link>http://biowww.net/forum/read/5/1065/1481#msg-1481</link>
      <author>charan</author>
      <description><![CDATA[Hi 
I am having issues with quick change mutagenesis kit.
I am trying to mutagenize a gene ,introduce one base substitution in a 2.5kb gene cloned into puc19. 
my oligos are 37base long with 18 base homology on either side
I follow exactly the same procedure as per the manual but only variation is that 
a) my oligos are not PAGE purified 
b) I do not use the competent cells that come with the kit
My issue is that I get NO colonies at all
I tried both chemical transformation and electroporation. But nothing seems to work.
IO saw someone with similar issues . My extension time is 1.5 min/kb and final extension is 10 min.
Can you suggest something. Also I  have no other option but plasmid mutagenesis. So has any one had any experience with other kits???????????/
Thanx
]]></description>
      <category>Molecular biology troubleshooting forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/5/1065/1481#msg-1481</guid>
      <pubDate>Tue, 27 Sep 2005 15:57:02 -0400</pubDate>
    </item>
    <item>
      <title>Re: Site directed mutagenesis</title>
      <link>http://biowww.net/forum/read/5/1065/1281#msg-1281</link>
      <author>rajabi</author>
      <description><![CDATA[I have plasmid which unfortunately occurs frameshift , of course framshift result from primer bad synthesis ( i  recommended  degenerate primer however both of nucleotide synthsis).

Plasmid= 4700 b.p.

Insert    =1200 b.p.

Additional two nucleotide in first gene

 please how deletion two nucleotide (simple methods).

Thank you very much for kind attention.

rajabi

 ]]></description>
      <category>Molecular biology troubleshooting forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/5/1065/1281#msg-1281</guid>
      <pubDate>Sun, 07 Aug 2005 05:50:15 -0400</pubDate>
    </item>
    <item>
      <title>Re: Site directed mutagenesis with PCR</title>
      <link>http://biowww.net/forum/read/5/1065/1220#msg-1220</link>
      <author>nit</author>
      <description><![CDATA[Hi Dan,
Thankyou very much. Just completed my work. My site directed mutagenesis worked with your conditions. Though I had troubles initially it finally worked and I confirmed the results also. Once again thank you very much.
]]></description>
      <category>Molecular biology troubleshooting forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/5/1065/1220#msg-1220</guid>
      <pubDate>Mon, 25 Jul 2005 00:56:41 -0400</pubDate>
    </item>
    <item>
      <title>Re: Site directed mutagenesis with PCR</title>
      <link>http://biowww.net/forum/read/5/1065/1094#msg-1094</link>
      <author>femmeauburn</author>
      <description><![CDATA[4 minutes may not be long enough for the extension step.  try increasing it to 10-15 minutes.]]></description>
      <category>Molecular biology troubleshooting forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/5/1065/1094#msg-1094</guid>
      <pubDate>Fri, 24 Jun 2005 09:02:19 -0400</pubDate>
    </item>
    <item>
      <title>Re: Site directed mutagenesis with PCR</title>
      <link>http://biowww.net/forum/read/5/1065/1086#msg-1086</link>
      <author>Dan</author>
      <description><![CDATA[Dear Nit,
 I realized that extension time is very important! you should  increase the extension time, I would do 10 minutes elongation time for your plasmid. And stick to the conditions reported on the manual 

1) 95 deg for 1 min
2)95 deg for 50 sec
3)58-60 deg for 50 sec
4)68 deg for 10 min
5)68 deg for 15 min
6) 4 deg infinite

repeat 2-4 for 18 times.

Try to increase the amount of template up to 50 ng and keep primer concentrations as indicated on the manual (125 ng each)

I hope this can help.
It worked for me and I wish you good luck...]]></description>
      <category>Molecular biology troubleshooting forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/5/1065/1086#msg-1086</guid>
      <pubDate>Wed, 22 Jun 2005 15:01:12 -0400</pubDate>
    </item>
    <item>
      <title>Re: Site directed mutagenesis with PCR</title>
      <link>http://biowww.net/forum/read/5/1065/1082#msg-1082</link>
      <author>nit</author>
      <description><![CDATA[My plasmid is only 3.7kb. Ya i am planning to introduce all the three sites at once. As i told earlier my primer is about 57 bases long and has a melting point of 87.5' C.  The conditions I have maintained for the PCR are same as that given in the Stratagene book. Conditions as follows:
1) 95 degree C for 30 sec
2) 95 degree C for 30 sec
3) 55 degree C for 1 min
4) 68 degree C for 4 min 
5) 4 degree for infinity time.
The steps 2-4 are repeated 18 times ( as given in the book for addition).
 Please let me know if I have to make any changes in the protocol
Thanks.              ]]></description>
      <category>Molecular biology troubleshooting forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/5/1065/1082#msg-1082</guid>
      <pubDate>Wed, 22 Jun 2005 01:51:09 -0400</pubDate>
    </item>
    <item>
      <title>Re: Site directed mutagenesis with PCR</title>
      <link>http://biowww.net/forum/read/5/1065/1080#msg-1080</link>
      <author>Dan</author>
      <description><![CDATA[I can only say I had several troubles to mutagenize  big plasmids and this gets much better increasing the elongation time (25 min for 10 KB) and slightly decreasing annealing temperature (58-59 deg). Are you planning to introduce three sites all at once??? Do you have primers long enough (at least 20 bp) on each side of the mismatch??
Precipitation of the PCR reaction and transformation of more DNA helps as well and I usually get transformants even if the PCR product is not detectable on gel.
Are your primers PAGE purified?
If you do not get the desired mutations, why do not try to introduce your mutation in sequent steps?

Good luck
]]></description>
      <category>Molecular biology troubleshooting forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/5/1065/1080#msg-1080</guid>
      <pubDate>Tue, 21 Jun 2005 18:13:51 -0400</pubDate>
    </item>
    <item>
      <title>Re: Site directed mutagenesis with PCR</title>
      <link>http://biowww.net/forum/read/5/1065/1077#msg-1077</link>
      <author>John H</author>
      <description><![CDATA[I mean the positive control provided by the kit. It may help you to determine if the problem araise from the pcr reaction, transformation etc other than primer design itself. The Tm you mentioned seems ok since the kit requires &gt;=78c. Any thoughts?]]></description>
      <category>Molecular biology troubleshooting forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/5/1065/1077#msg-1077</guid>
      <pubDate>Tue, 21 Jun 2005 09:29:19 -0400</pubDate>
    </item>
    <item>
      <title>Re: Site directed mutagenesis with PCR</title>
      <link>http://biowww.net/forum/read/5/1065/1076#msg-1076</link>
      <author>nit</author>
      <description><![CDATA[Hi John
Thanks. But what exactly do you mean by control? I did keep a control of only DNA and water mixture for PCR and was unable to get any colonies. So I prepared fresh plasmid DNA by miniprep kit. Can you please suggest me the conditions to be maintained for this site directed mutagenesis as my primer melting point are 87.5' C.]]></description>
      <category>Molecular biology troubleshooting forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/5/1065/1076#msg-1076</guid>
      <pubDate>Tue, 21 Jun 2005 02:23:29 -0400</pubDate>
    </item>
    <item>
      <title>Re: Site directed mutagenesis with PCR</title>
      <link>http://biowww.net/forum/read/5/1065/1075#msg-1075</link>
      <author>John H</author>
      <description><![CDATA[Did you get any colonies with the control reaction? Ensure the control is working and then check if the primer design is correct.]]></description>
      <category>Molecular biology troubleshooting forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/5/1065/1075#msg-1075</guid>
      <pubDate>Mon, 20 Jun 2005 20:04:11 -0400</pubDate>
    </item>
    <item>
      <title>Site directed mutagenesis with PCR</title>
      <link>http://biowww.net/forum/read/5/1065/1065#msg-1065</link>
      <author>nit</author>
      <description><![CDATA[Hi
I want to introduce three enzyme sites and an RBS site in my plasmid. So i decided to carry out site directed mutagenesis wherein with one pcr I would be able to introduce all the sites. I designed the forward and reverse preimers correspondingly. But now i am not too sure about the condition i should maintain during my pcr. I tried with the standard protocol given in the stragene kit (which i used) but i dont think it worked. The melting point of my primer is 87.5' C. Also I am not able to get any colonies on transformation.
It would be great if some one could point out the condition and the error. I need to be done with my work in a few weeks time.
Any thought will be greatly appreciated.
Thanks...
]]></description>
      <category>Molecular biology troubleshooting forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/5/1065/1065#msg-1065</guid>
      <pubDate>Sun, 19 Jun 2005 04:32:32 -0400</pubDate>
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