Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Site-directed mutagenesis failed


  • Please log in to reply
20 replies to this topic

#1 Shifting Reality

Shifting Reality

    member

  • Active Members
  • Pip
  • 24 posts
0
Neutral

Posted 10 December 2013 - 02:25 AM

hello everybody,

 

let me share my frustration :)

 

i would like to make a site-directed mutagenesis on a vector but it has been failed again and again.

 

I mean I have never got the amplified mutant plasmid.

 

here is my protocol:

 

- x ul plasmid template (50 ng)

- 1 ul FW primer (10 pmol/ul)

- 1 ul REV primer (10 pmol/ul)

fully complementer to each other (36 bp , Tm: 83 C)

- 5 ul buffer (10X) [buffer contains Mg2+]

- 1 ul Pfu ultra II polymerase

- 1,5 ul dNTP mix (10 mM)

- x ul H2O

 

Vt: 50 ul

- - -

denaturing temperature: 95 C

annealing temperature: 55 C

extension temperature: 72 C

25 cycles

 

I don't know the "hidden factor" that wasn't to be considered...

 

SF



#2 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,484 posts
251
Excellent

Posted 10 December 2013 - 06:50 AM

You don't say what the result is. Do you get transformants that are original vector? Do you get no transfomants?

 

I'd say you should dramatically lower the template concentration. After PCR, you should digest the DNA product with DpnI to eliminate template DNA. Both of these will help you suppress transformed cells with the original plasmid sequence.



#3 Shifting Reality

Shifting Reality

    member

  • Active Members
  • Pip
  • 24 posts
0
Neutral

Posted 10 December 2013 - 07:59 AM

Hello phage,

 

I didn't perform the DpnI digestion then the transformation cause I didn't get PCR product. That's the basic problem.



#4 student47

student47

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 46 posts
3
Neutral

Posted 10 December 2013 - 09:35 AM

i have a feeling my suggestion could be wrong but here it is.

check if its the right plasmid. primer/sequence.

some inhibitory factor in one of the extracts is inhibiting the reaction possibly.

i usually use 34 cycles

do you have any primer dimers?

what do you mean by complete complimentarity.?

use a different polymerase or and buffer.

hope u resolve it.



#5 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,484 posts
251
Excellent

Posted 10 December 2013 - 04:04 PM

I assume the mutation you are attempting to insert is n the middle of the 36 bp. How confident are you that the plasmid contains the matching sequence (except for the mutation you need to insert)? How are you determining that your PCR fails to work? With 50 ng of template, you will always get a band at the correct length.



#6 Shifting Reality

Shifting Reality

    member

  • Active Members
  • Pip
  • 24 posts
0
Neutral

Posted 11 December 2013 - 07:54 AM

Thank you for your comments, guys ! It is sure that the plasmid contains the matching sequence - I have sequence results. I thought that PCR didn't work cause I only saw the template plasmid at the correct lenght. I assume if I have a successful PCR reaction the result is a very bright band not the template band of 50 ng DNA.

 

Do you think that primer concentration is fine (10 pmol/ul) ?



#7 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,784 posts
406
Excellent

Posted 11 December 2013 - 12:55 PM

For SDM protocols the PCR step is NOT an amplification step, it is about replicating the plasmid DNA so that you can then digest away the methylated parent DNA. 

 

In other words - Do the DpnI digest....



#8 student47

student47

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 46 posts
3
Neutral

Posted 11 December 2013 - 03:04 PM

hi this answer by bob 1 makes sense..

 

For SDM protocols the PCR step is NOT an amplification step, it is about replicating the plasmid DNA so that you can then digest away the methylated parent DNA. 

 

In other words - Do the DpnI digest....



#9 Shifting Reality

Shifting Reality

    member

  • Active Members
  • Pip
  • 24 posts
0
Neutral

Posted 13 December 2013 - 06:13 AM

Thanks guys ! It really makes sense. What a stuped I am :D



#10 Shifting Reality

Shifting Reality

    member

  • Active Members
  • Pip
  • 24 posts
0
Neutral

Posted 01 May 2014 - 07:02 AM

Hello everbody,

 

I would like to know opinions about my question: is it possible to make SDM with long primers (50-100 nt) that have high Tm ? Tm 90 or 100 C primers, for example. With a relatively high annealing temperature and the use of DMSO for avoiding secondary structure of the long primers.

 

SR



#11 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,784 posts
406
Excellent

Posted 01 May 2014 - 01:10 PM

How similar are the primers to the target sequence?  Any bases that aren't supposed to anneal should be discounted from the annealing temp calculations.



#12 Shifting Reality

Shifting Reality

    member

  • Active Members
  • Pip
  • 24 posts
0
Neutral

Posted 02 May 2014 - 02:13 AM

There are insertions and substitutions in the primer. The annealed parts of the primers have 97,6 C Tm. The whole primers have 100 C Tm. Not too different...



#13 Trof

Trof

    Brain on a stick

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,201 posts
109
Excellent

Posted 02 May 2014 - 02:56 AM

Primers actually never get such high Tm.

The Tm is increasing with length, but only to certain point, then it stalls. Because such long sequence doesn't melt at single point anymore, but creates smaller melting domains. Software that calculates Tm doesn't take this usually into account, because it's not that predictable.

When you have long primers, they even aren't more specific. From a length around 25-30 bp the specificity doesn't increase anymore, because it's so long, that only 3' end of primers anneal (this is used when designing long primers with 5' noncomplementary sequence like a restriction site, you can just add the aditional bp to a normal primer and use the Ta as you would for the primer alone, the 5' end will just hang freely).

 

So, too long primers have no additional benefits (SDM primers have around 30, because they have an inside mismatch and both ends have to be long enough to compensate for the mispriming to be specific enough, but no more) but only more and more problems with secondary structure.

 

Also it's not commonly available to sythesize such long primers, or/and the prices are much higher.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#14 Shifting Reality

Shifting Reality

    member

  • Active Members
  • Pip
  • 24 posts
0
Neutral

Posted 03 May 2014 - 07:00 AM

Ok. Thanks for your comments !



#15 labtastic

labtastic

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 78 posts
2
Neutral

Posted 06 May 2014 - 11:45 AM

If a quikchange mutagenesis reaction doesn't work the first time, I just go straight to overlap extension to make the mutation. I have found myself spending days trying to troubleshoot a quickchange reaction when I could have just done the overlap extension, which although a bit more involved than quickchange works beautifully and every colony will have your mutation. Plus you can do multiple mutations at once with overlap extension as long as they are far enough apart in sequence (>100bp....though if the mutations are less than that far apart you can just have multiple mutations in a single primer). When amplifying for overlap extension, I have had great success with touchdown PCR as well- nice clean single bands every time.  Just my two cents.  






Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.