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Cloning trouble


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#1 Ecoli0157

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Posted 18 February 2009 - 08:05 AM

I have cloned a range of g.DNA digests (from 2~4k.b.) into pUC19, some of which should be the desired inserts that include transposon and some flanking regions. I did a total of 20 transformations (heatshock) and spun down the cells and spread the concentrated cells onto LB/Kan(Transposon contains Kan resistant marker). My controls all looked fine, but a few mysterious results.

1) I got some colonies which began to show up after 2~3 days of incubation and by 5th day they were still pretty tiny.

2) These colonies were re-streaked onto LB, LB/Kan, LB/Kan and Amp.
No growth so far after one day incubation on any of these
I began to see some growing on LB/kan upon 2 day of incubation

Also, broth inoculation (LB/ Kan&Amp) also shows no growth after two day of incubation.

Has anyone experienced something like this?
I am not sure what could be giving resistance to Kan because it is obvious that only transposon fragment ligated with pUC19 should generate kan and amp resistant clones.

Another note is that comp. cell used was DH5alpha which is recA- so I don't think any kind of recombination occured.

If anyone can give me suggestions on what might have happend and what I should do, I would humbly accept them

Thanks,

#2 AquaPlasmid

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Posted 18 February 2009 - 09:48 AM

I had your experience with DH5a and kan selection. Those needle size colonies don't have plasmid and unlike amp satellites they don't grow up in liquid cultures with or without antibiotics. Never dug into it to understand why though.

#3 perneseblue

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Posted 18 February 2009 - 10:56 AM

There are no plasmids in your cells. Even though DH5a grows slowly, more than 2 days is past the acceptable limit.

Could you tell us about the digest and ligation between the genomic DNA fragments and the pUC19 vector? How did the digest go? Have there been problems with the ligase or ligase buffer (the ligase buffer should smell strongly of DTT)? The both components but the T4 ligase especially is prone to going off.
May your PCR products be long, your protocols short and your boss on holiday

#4 Ecoli0157

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Posted 18 February 2009 - 11:28 AM

There are no plasmids in your cells. Even though DH5a grows slowly, more than 2 days is past the acceptable limit.

Could you tell us about the digest and ligation between the genomic DNA fragments and the pUC19 vector? How did the digest go? Have there been problems with the ligase or ligase buffer (the ligase buffer should smell strongly of DTT)? The both components but the T4 ligase especially is prone to going off.


Thanks so much for your answer!

I set up 12 separate overnight digests and they all produced a uniform smear on the gel, probably indicating that digests went to completion (Especially, when EcoR1 is a pretty robust enzyme). I did a control ligation with non-CIP treated vector and this vector was re-ligated and transformed. I had a good number of colonies on LB/amp plates.T4 DNA Ligase was close to the expiration date, but not that old and also Ligase buffer has been aliqouted into small volumes. Therefore, I concluded that ligation condition was good enough and the cut and re-ligated vector really did re-ligate.
If things are not working out, is it reasonable to run your ligations on the gel before transformation because I normally dont do that.
Also, I learned that CIP is a tough enzyme to get rid of. I used spin-column purification, but I might do a phenol-chloro purification or even just do it without CIP treating the vector and play with the Ins:vect ratios.
Electroporation rather than heat shock method might give better results too!

#5 Ecoli0157

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Posted 18 February 2009 - 11:31 AM

I had your experience with DH5a and kan selection. Those needle size colonies don't have plasmid and unlike amp satellites they don't grow up in liquid cultures with or without antibiotics. Never dug into it to understand why though.


Thanks for the reply. It is exactly what I am getting. Some kind of "ghost colonies".
Actually, could you tell me what you did differently to get your clone then?
(Did you change competent cell or vector?)

I have Amp marker from vector and Kan from the insert. I was wondering if this is too much stress for the recombinant clones if they are exposed both antiobiotics following 1 hour of incubation in SOC medium.

Edited by Ecoli0157, 18 February 2009 - 12:04 PM.


#6 perneseblue

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Posted 18 February 2009 - 01:19 PM

If things are not working out, is it reasonable to run your ligations on the gel before transformation.

Very reasonable. I would recommend that you do this test for the next ligation.

As for CIP, also note that over dephosphorylation can damage the end of your vector, leaving the vector unligatable. So keep to the dephos instructions. A control for this would be CIP vector + polynucleotide kinase (PNK) + t4 ligase. This should give high transformation frequency. If it doesn't the vector has been damaged.

As for removal of CIP, I use phenol. I agree CIP is a tough enzyme it can't be heat inactivated.

Electroporation rather than heat shock method might give better results too!

Electroporation most definitely gives better transformation yields.

I have Amp marker from vector and Kan from the insert. I was wondering if this is too much stress for the recombinant clones if they are exposed both antiobiotics following 1 hour of incubation in SOC medium.

The bacteria should be okay.
May your PCR products be long, your protocols short and your boss on holiday




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