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
- - - - -

SM10 lambda pir covers my plates


  • Please log in to reply
4 replies to this topic

#1 cerqueiragm

cerqueiragm

    member

  • Active Members
  • Pip
  • 6 posts
0
Neutral

Posted 26 March 2011 - 06:39 PM

Hi everybody,
today I come to you with an interesting challenge! Prepare your brains, take your notes and GO!

I have been working with the HIGH COPY NUMBER suicide vector pSSK10 and trying to clone two fragments within it, simultaneously. I made a very good plasmid preparation, linearized very well (double digestion using NdeI and XhoI), dephosphorilated using CIP and cut from the gel. So far, everything was perfect.

For cloning I used, as a reference, 100 ng of the linear vector. Both fragments I want to clone contain 1500 bp, which correspond to the flanks of the gene I want to further mutate.

I runned my ligations at 25C for a few hours and transformed 100 uL of E. coli SM10 lambda Pir with the whole 15 microliters of my ligation. Then, I've plated 10% of the 2YT resuspended transformation and the remaining amount onto HI/Kan plates.

While for most of the vectors and E. coli lineages I've worked in the past this would result in a rasonable number of colonies for further screening, for SM10 lambda pir transformed with such ligation mix I've got a true bacterial lawn. The next days plates were competely covered and no single colonies could be distinguished.

An important detail... It happened twice. I've used different SM10 lambda-pir chemically competent cells sources, from two different labs (So I know it is not cells fault), and I used different vector batches for cloning (so I know it is not bad preparation or contamination with circular vector).

Here it goes my challenging question that will make you think for the next few hours... what is going on? What I should do to obtain isolated colonies onto my next day plates and still ensure the obtaining of recombinant clones?

Thanks a lot guys. I'll try your suggestions and the first to help me obtaining success I'll give an Amazon.com gift card of U$ 10,00. My way to say thank you.
See you.
Gustavo.

Edited by cerqueiragm, 26 March 2011 - 06:42 PM.


#2 pDNA

pDNA

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 491 posts
14
Good

Posted 26 March 2011 - 11:43 PM

you did proper controls to check if your Kan plates are working?
this means plating the competent cells transformed with sterile water ...maybe the Kan in your plates is degraded?
What concentration do you use for selection?

you mentioned that you are sure that it is not caused by a bad preparation or contamination with circular vector due to the fact that you tried two different batches of vector preperations. This seems to be a weak argument to me. What if one of your restriction enzymes is dead for example?

I would streak cells from your lawn onto a fresh Kan plate to get single colonies and would try to do a plasmid isolation to check what confers the resistance. Maybe its really some background. And if you are not able to isolate a plasmid then something is wrong with your plates or cells.

Regards,
p

Hi everybody,
today I come to you with an interesting challenge! Prepare your brains, take your notes and GO!

I have been working with the HIGH COPY NUMBER suicide vector pSSK10 and trying to clone two fragments within it, simultaneously. I made a very good plasmid preparation, linearized very well (double digestion using NdeI and XhoI), dephosphorilated using CIP and cut from the gel. So far, everything was perfect.

For cloning I used, as a reference, 100 ng of the linear vector. Both fragments I want to clone contain 1500 bp, which correspond to the flanks of the gene I want to further mutate.

I runned my ligations at 25C for a few hours and transformed 100 uL of E. coli SM10 lambda Pir with the whole 15 microliters of my ligation. Then, I've plated 10% of the 2YT resuspended transformation and the remaining amount onto HI/Kan plates.

While for most of the vectors and E. coli lineages I've worked in the past this would result in a rasonable number of colonies for further screening, for SM10 lambda pir transformed with such ligation mix I've got a true bacterial lawn. The next days plates were competely covered and no single colonies could be distinguished.

An important detail... It happened twice. I've used different SM10 lambda-pir chemically competent cells sources, from two different labs (So I know it is not cells fault), and I used different vector batches for cloning (so I know it is not bad preparation or contamination with circular vector).

Here it goes my challenging question that will make you think for the next few hours... what is going on? What I should do to obtain isolated colonies onto my next day plates and still ensure the obtaining of recombinant clones?

Thanks a lot guys. I'll try your suggestions and the first to help me obtaining success I'll give an Amazon.com gift card of U$ 10,00. My way to say thank you.
See you.
Gustavo.



#3 Benjamin Simon

Benjamin Simon

    member

  • Members
  • Pip
  • 1 posts
1
Neutral

Posted 12 May 2011 - 08:36 AM

As far as I know, SM10 Lambda pir is Kanamycin resistant. The genotype lists "Km". Use a Step resistant suicide plasmid like pKNG101.

#4 ooztas

ooztas

    member

  • Members
  • Pip
  • 1 posts
-1
Neutral

Posted 27 May 2011 - 04:17 AM

Hi Gustavo,
Have you solved your problem? I have also the same problem with SM10 cloning. I am using pGP704-SacB low copy suicide vector and trying to clone the construct including chloramphenicol resistance gene. When I selected the cells on ampicillin plate, it results smear. Are you sure that your cells are SM10. Because when I controlled my SM10, it grows on kanamycin,ampicillin and chloramphenicol and not on streptomycin. The shape is also similar with SM10, however it is not SM10.

#5 cerqueiragm

cerqueiragm

    member

  • Active Members
  • Pip
  • 6 posts
0
Neutral

Posted 14 June 2011 - 06:22 PM

Hi Benjamin,
sorry for my delay to reply back to you. You're right and I noticed that after double checking the genotype for the strains I have been working with. Please, provide me an email address to send you the gift card. It can be used for shopping in Amazon.com.
Thx a lot for your help.
Best regards.




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

©1999-2013 Protocol Online, All rights reserved.