Methods Digest, Vol 42, Issue 18

Mahesh Reddy via methods%40net.bio.net (by vasipalli from me.com)
Sat Nov 29 17:07:59 EST 2008


I prefer to use XL-10 gold competent cells.you could get from  
stratagene.

Sent from my iPhone

On 29 Nov 2008, at 17:04, methods-request from oat.bio.indiana.edu wrote:

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> Today's Topics:
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>   1. Efficiency of (electro)transformation in Salmonella (C Coward)
>   2. Re: Efficiency of (electro)transformation in Salmonella (DK)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: 28 Nov 2008 16:33:34 GMT
> From: C Coward <cc122 from mole.bio.cam.ac.uk>
> Subject: Efficiency of (electro)transformation in Salmonella
> To: methods from net.bio.net
> Message-ID: <ggp6gu$qo2$1 from gemini.csx.cam.ac.uk>
>
> Hello all,
>
> I'm after opinions/advice on how to get the best transformation  
> efficiency
> in Salmonella (enteritidis).
>
> I'm currently using electroporation but am routinely getting  
> efficiencies
> around 10^4 cf/ug of DNA. I don't think restriction systems would be
> reducing my efficiency as the plasmid DNA is prepared from a  
> Salmonella
> strain (I haven't tested this though, the plasmid DNA is coming from  
> S.
> typhimurium LB5010). I do need to test the efficiency of re- 
> introducing my
> plasmid preps into LB5010.
>
> I could really do with higher efficiency so any tips?
>
> My protcol is as follows
>
> 1. Grow a starter O/N in LB from 37C
> 2. Inoculate 20ml with 100ul of the starter, grow @37C to OD~0.5
> 3. Wash 1X in 20ml ice-cold dH2O
> 4. Pellet and wash 1X in 1ml ice-cold dH2O
> 5. Pellet and wash 2X in 1ml ice-cold 10% glycerol
> 6. Resuspend in 400ul 10% glycerol, aliquot in 100ul lots, store at  
> -80
> 7. Electroporate using 2mm gap cuvettes, 2kV, 25uF, 200ohm (pulse time
> around 5ms)
>
> Cheers!
>
>
>
> ------------------------------
>
> Message: 2
> Date: Fri, 28 Nov 2008 17:11:35 GMT
> From: dk from no.email.thankstospam.net (DK)
> Subject: Re: Efficiency of (electro)transformation in Salmonella
> To: methods from net.bio.net
> Message-ID: <aDVXk.606$Re2.528 from newsfe14.iad>
>
> In article <ggp6gu$qo2$1 from gemini.csx.cam.ac.uk>, C Coward <cc122 from mole.bio.cam.ac.uk 
> > wrote:
>> Hello all,
>>
>> I'm after opinions/advice on how to get the best transformation  
>> efficiency
>> in Salmonella (enteritidis).
>>
>> I'm currently using electroporation but am routinely getting  
>> efficiencies
>> around 10^4 cf/ug of DNA. I don't think restriction systems would be
>> reducing my efficiency as the plasmid DNA is prepared from a  
>> Salmonella
>> strain (I haven't tested this though, the plasmid DNA is coming  
>> from S.
>> typhimurium LB5010). I do need to test the efficiency of re- 
>> introducing my
>> plasmid preps into LB5010.
>>
>> I could really do with higher efficiency so any tips?
>>
>> My protcol is as follows
>>
>> 1. Grow a starter O/N in LB from 37C
>> 2. Inoculate 20ml with 100ul of the starter, grow @37C to OD~0.5
>> 3. Wash 1X in 20ml ice-cold dH2O
>> 4. Pellet and wash 1X in 1ml ice-cold dH2O
>> 5. Pellet and wash 2X in 1ml ice-cold 10% glycerol
>> 6. Resuspend in 400ul 10% glycerol, aliquot in 100ul lots, store at  
>> -80
>> 7. Electroporate using 2mm gap cuvettes, 2kV, 25uF, 200ohm (pulse  
>> time
>> around 5ms)
>
> 1. Try higher cell concentration. Typical E.coli preps are 1 L  
> culture -->
> resuspend in 2 ml final, i.e. 500X. Yours is only 50X.
> 2. Vary voltage. E.g. 1.8, 2.2, 2.4 kV for 2 mm gap, 1.4,1.6,1.8 kV
> for 1 mm gap.
> 3. Try 7% and 10% DMSO in place of glycerol.
> 4. Try growing cultures at 20C instead of 37. Gives a 10X boost
> for E.coli.
> 5. Try including 50 mM DTT in washes (will require voltage
> optimization since when  the cell wall is weakened the electric
> filed intensity needs to be reduced).
>
> DK
>
>
>
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>
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> End of Methods Digest, Vol 42, Issue 18
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