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Colony Screeing without using PCR


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#1 Timothy007

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Posted 05 February 2010 - 08:24 AM

Does anyone know how to quickly screen multiple recombinant colonies for "properly oriented" insert from agar plate by restriction enzyme digestion instead of commonly used(expensive) PCR method?
I know some of the (expensive)kit can perform the job by:

1. Picking the colony with toothpick
2. Addition of proprietary lysis buffer
3. Boiling
4. Restriction analysis.

What are the actual components of the lysis buffer?
Can this method work with HB101 series E.coli strains?
Any other method?

#2 leelee

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Posted 06 February 2010 - 03:43 AM

I would have thought that RE digest screening would be much the same price as PCR, if not more expensive.

#3 HomeBrew

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Posted 06 February 2010 - 06:36 AM

See the "Restriction enzyme digestion-based screening" section here.

#4 Timothy007

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Posted 06 February 2010 - 04:24 PM

See the "Restriction enzyme digestion-based screening" section here.



I tried with JM101 using EB for staining, it dosen't work.

Has anyone tried this method or others?

Edited by Timothy007, 06 February 2010 - 04:28 PM.


#5 HomeBrew

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Posted 06 February 2010 - 07:31 PM

It's probably not so much the E. coli strain, but the vector. What plasmid are you cloning into?

#6 Timothy007

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Posted 06 February 2010 - 07:46 PM

It's probably not so much the E. coli strain, but the vector. What plasmid are you cloning into?

pBR322 coE1 replicator

#7 HomeBrew

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Posted 07 February 2010 - 05:32 AM

Plasmids based on the ColE1 origin of pBR322 are extremely low copy number plasmids -- about 10-20 per cell (as opposed to pMB1-based plasmid like the pUC series, which can exist in copy numbers approaching 1000 per cell). This is most likely what your problem is -- it's not that the protocol "doesn't work", it's that the plasmid yield from the amount of cells you're trying to use is insufficient for visualization on an EtBr-stained gel.

#8 Timothy007

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Posted 07 February 2010 - 06:12 AM

Plasmids based on the ColE1 origin of pBR322 are extremely low copy number plasmids -- about 10-20 per cell (as opposed to pMB1-based plasmid like the pUC series, which can exist in copy numbers approaching 1000 per cell). This is most likely what your problem is -- it's not that the protocol "doesn't work", it's that the plasmid yield from the amount of cells you're trying to use is insufficient for visualization on an EtBr-stained gel.


But I also tried with another plasmid based on pUC18. It dosen't work also 囧....

Edited by Timothy007, 07 February 2010 - 06:15 AM.


#9 HomeBrew

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Posted 07 February 2010 - 01:03 PM

Ok -- so you:

  • Picked colonies with a 200 ul pipette tip and dispersed them into tubes containing 50 ul culture medium with antibiotic.
  • Pipetted 3-4 times to re-suspend the colonies.
  • Incubated the mini-cultures at 37 deg for 2-4 hours with aeration.
  • Combined 20 ul the mini-cultures with 20 ul of 2x direct lysis buffer.
  • Vortexed the suspensions, then boiled them for 90 secs.
  • Centrifuged at high speed (12k-20k g) at room temperature for 10 min and collected supernatants.
  • Used all (40 ul) of the collected supernatants for restriction enzyme digestion.
  • Loaded the total digest on a gel.

and saw nothing?

If that's the case, I would add a 37 deg incubation for 10 minutes after combining the 20 ul the mini-cultures with 20 ul of 2x direct lysis buffer to give the lysozyme a chance to work, then proceed.




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