Methods Digest, Vol 21, Issue 25

[Jayanta Tarafdar]

Dear all,
   
  Anybody, please suggests the protocol (modfied, if any) of DNA extarction from chilli leaves, We are using CTAB method but bands of cut & uncut DNA are coming foolish.
   
  Jayanta 

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Today's Topics:

1. Re: Pipetting, contamination.... (DK)
2. Re: Pipetting, contamination.... (DK)
3. Re: Pipetting, contamination.... (peter)
4. Re: Pipetting, contamination.... (peter)
5. Re: Pipetting, contamination.... (jg374 from cam.ac.uk)
6. Re: Pipetting, contamination.... (Christian Praetorius)
7. Re: Pipetting, contamination.... (Christian Praetorius)
8. Re: "PCR Methods and Applications" (Christian Praetorius)
9. Re: Pipetting, contamination.... (ChenHA)
10. Re: Purifying PCR product from 2% agarose gel (ChenHA)


----------------------------------------------------------------------

Message: 1
Date: Fri, 23 Feb 2007 03:26:25 GMT
From: dk from no.email.thankstospam.net (DK)
Subject: Re: Pipetting, contamination....
To: methods from net.bio.net
Message-ID: 

In article <1172199429.419560.225640 from p10g2000cwp.googlegroups.com>, "peter" 
wrote:
>On Feb 22, 9:09 pm, d... from no.email.thankstospam.net (DK) wrote:
>I guess we will find out.... sometimes bacteria will fail to grow if
>you seed directly to >100 ml (even if you pick from a colony), that's
>why people make small culture overnight.

Nope, people make small cultures overnight to make sure they've got 
saturated cultures no matter how much of the cells they inoculated, 
certainly not because bacterial cells die when diluted. 

Having uniform saturated cultures can offer various advantages
such as a) inoculating larger cultures with ~ the same amounts
so that the desired density is reached ~ at the same time, b) having 
maximal amount of plasmid per cell. 

Inoculating into larger volumes will only increase lag phase, but with the 
difference between 2 ml and 100 ml of only 50X ~ 6 doublings, I can't 
see how it would make an all ot none difference. An eye easily picks 
turbidity at as low as OD600=0.05. 

DK


------------------------------

Message: 2
Date: Fri, 23 Feb 2007 05:58:29 GMT
From: dk from no.email.thankstospam.net (DK)
Subject: Re: Pipetting, contamination....
To: methods from net.bio.net
Message-ID: 

In article <1172202357.518158.176980 from 8g2000cwh.googlegroups.com>, "peter" 
wrote:
>On Feb 22, 10:26 pm, d... from no.email.thankstospam.net (DK) wrote:

>> Inoculating into larger volumes will only increase lag phase, but with the
>> difference between 2 ml and 100 ml of only 50X ~ 6 doublings, I can't
>> see how it would make an all ot none difference. An eye easily picks
>> turbidity at as low as OD600=0.05.
>>
>> DK
>
>DK , I did not say that bacteria die, I said it can fail to grow -
>especially if you have ampicillin, which is bacteriostatic.

Sure. But, naturally, I did not have any antibiotic, just plain rich 
medium. So knowing what we know about E.coli, I don't see how 
it would fail to grow. You know, a single colony on a plate does 
grow - even though it was just one cell per whole plate... 

Personally, I think this is a case of failing to find black cat in the 
dark room when the cat is not there. Like I said earlier in this 
thread: aerosol contamination is highly overrated. 

DK





------------------------------

Message: 3
Date: 22 Feb 2007 18:57:09 -0800
From: "peter" 

Subject: Re: Pipetting, contamination....
To: methods from net.bio.net
Message-ID: <1172199429.419560.225640 from p10g2000cwp.googlegroups.com>
Content-Type: text/plain; charset="iso-8859-1"

On Feb 22, 9:09 pm, d... from no.email.thankstospam.net (DK) wrote:
> In article <1172192382.552109.275... from l53g2000cwa.googlegroups.com>, "peter" 
wrote:
>
>
>
> >On Feb 22, 7:41 pm, d... from no.email.thankstospam.net (DK) wrote:
> >> In article <1172181256.088647.198... from a75g2000cwd.googlegroups.com>, "peter"
> > 
wrote:
>
> >> >> Jeez, I've done this experiment yesterday:
> >> >> 1. Took a 1 ml Pipetman, set it to 1 ml and pipetted overnight E.coli
> >> >> culture up and down 20 times.
> >> >> 2. Immediately after that, changed a tip, took a bottle of sterile
> >> >> Terrific Broth, pipetted 20 times, set at 37C for 20 hours.
>
> >> >> The result: no signs of bacterial growth today ==> no aerosol
> >> >> contamination.
>
> >> >> DK
>
> >> >Seems like a good experiment, I just wonder what volume of the TB
> >> >media you used?
>
> >> The standard square bottle filled with 100 ml. I still have that bottle,
> >> so if you have your doubts, I can look at it very attentively by Monday.
> >> But, you know, E.coli can grow anaerobically so shaking is not
> >> strictly necessary...
>
> >> I pretty much knew the outcome based on the fact that I am still
> >> using the same ~1.5 years old bottle of SOC used for an umpteen
> >> (more like ~ 100 :-)) electroporations, each involving the same
> >> 1 ml Pipetman and no filter tips all along. Amazingly, still no visible
> >> growth in the bottle and, as recently as two days ago, 20 out of 20
> >> clones analyzed contained the expected plasmid.
>
> >> DK
>
> >> P.S. This topic reminded me of one curious observation:
>
> >> Everyone I know who does "microbiology" outside of sterile hood
> >> is engaged in the practice of flaming necks of every bottle and
> >> flask they use. When asked why they are doing it, the only answer
> >> ever given is "to keep it sterile". Problem is, for as far as I can tell,
> >> the way it is done, it's a completely useless exercise in at least
> >> 9 out of 10 times.
>
> >DK ... People make night cultures for a reason before they seed 100
> >ml botles. wonder why? Can you repeat same experiment with like 2 ml
> >TB or SOC?
>
> I can but what's the difference from looking at the bottle 3 days later?
>
> DK

I guess we will find out.... sometimes bacteria will fail to grow if
you seed directly to >100 ml (even if you pick from a colony), that's
why people make small culture overnight.



------------------------------

Message: 4
Date: 22 Feb 2007 19:45:57 -0800
From: "peter" 

Subject: Re: Pipetting, contamination....
To: methods from net.bio.net
Message-ID: <1172202357.518158.176980 from 8g2000cwh.googlegroups.com>
Content-Type: text/plain; charset="iso-8859-1"

On Feb 22, 10:26 pm, d... from no.email.thankstospam.net (DK) wrote:
> In article <1172199429.419560.225... from p10g2000cwp.googlegroups.com>, "peter" 
wrote:
>
> >On Feb 22, 9:09 pm, d... from no.email.thankstospam.net (DK) wrote:
> >I guess we will find out.... sometimes bacteria will fail to grow if
> >you seed directly to >100 ml (even if you pick from a colony), that's
> >why people make small culture overnight.
>
> Nope, people make small cultures overnight to make sure they've got
> saturated cultures no matter how much of the cells they inoculated,
> certainly not because bacterial cells die when diluted.
>
> Having uniform saturated cultures can offer various advantages
> such as a) inoculating larger cultures with ~ the same amounts
> so that the desired density is reached ~ at the same time, b) having
> maximal amount of plasmid per cell.
>
> Inoculating into larger volumes will only increase lag phase, but with the
> difference between 2 ml and 100 ml of only 50X ~ 6 doublings, I can't
> see how it would make an all ot none difference. An eye easily picks
> turbidity at as low as OD600=0.05.
>
> DK

DK , I did not say that bacteria die, I said it can fail to grow -
especially if you have ampicillin, which is bacteriostatic.
my2c



------------------------------

Message: 5
Date: Fri, 23 Feb 2007 09:54:46 +0000
From: "jg374 from cam.ac.uk" 
Subject: Re: Pipetting, contamination....
To: methods from net.bio.net
Message-ID: 

Content-Type: TEXT/PLAIN; charset=US-ASCII; format=flowed

On Fri, 23 Feb 2007, DK wrote:
> In article <1172181256.088647.198980 from a75g2000cwd.googlegroups.com>, "peter" 
wrote:
>>
>>> Jeez, I've done this experiment yesterday:
>>> 1. Took a 1 ml Pipetman, set it to 1 ml and pipetted overnight E.coli
>>> culture up and down 20 times.
>>> 2. Immediately after that, changed a tip, took a bottle of sterile
>>> Terrific Broth, pipetted 20 times, set at 37C for 20 hours.
>>>
>>> The result: no signs of bacterial growth today ==> no aerosol
>>> contamination.
>>>
>>> DK
>>
>> Seems like a good experiment, I just wonder what volume of the TB
>> media you used?
>
> The standard square bottle filled with 100 ml. I still have that bottle,
> so if you have your doubts, I can look at it very attentively by Monday.
> But, you know, E.coli can grow anaerobically so shaking is not
> strictly necessary...
>
> I pretty much knew the outcome based on the fact that I am still
> using the same ~1.5 years old bottle of SOC used for an umpteen
> (more like ~ 100 :-)) electroporations, each involving the same
> 1 ml Pipetman and no filter tips all along. Amazingly, still no visible
> growth in the bottle and, as recently as two days ago, 20 out of 20
> clones analyzed contained the expected plasmid.

I coughed into a bottle of SOC a few months ago as an experiment and it 
still looks fresh, however I have seen horrible things growing in SOC 
bottles that have supposedly only been opened under careful flaming.


------------------------------

Message: 6
Date: Fri, 23 Feb 2007 10:11:08 +0000
From: Christian Praetorius 

Subject: Re: Pipetting, contamination....
To: methods from net.bio.net
Message-ID: <547stkF1uufhtU1 from mid.individual.net>
Content-Type: text/plain; charset=us-ascii

dk from no.email.thankstospam.net (DK) wrote:

>Everyone I know who does "microbiology" outside of sterile hood 
>is engaged in the practice of flaming necks of every bottle and 
>flask they use. When asked why they are doing it, the only answer 
>ever given is "to keep it sterile". Problem is, for as far as I can tell,
>the way it is done, it's a completely useless exercise in at least 
>9 out of 10 times. 

Thats also my experience. In many cases the turbulences caused by the
flame disturbe the air and can cause contamination. A former colleague
of mine is is microbiologist, and they tested how long standard glas
pipettes stay sterile in the standard laboratory athmosphere. They
opened the autoclaved container, and it took them minimum two weeks
before this pipettes couldn't used any more because they were not
sterile any more.

Christian

-- 
X-no-Sig: yes


------------------------------

Message: 7
Date: Fri, 23 Feb 2007 10:13:01 +0000
From: Christian Praetorius 

Subject: Re: Pipetting, contamination....
To: methods from net.bio.net
Message-ID: <547t15F1uufhtU2 from mid.individual.net>
Content-Type: text/plain; charset=us-ascii

dk from no.email.thankstospam.net (DK) wrote:

>thread: aerosol contamination is highly overrated. 

If people work properly and clean.

Christian

-- 
X-no-Sig: yes


------------------------------

Message: 8
Date: Fri, 23 Feb 2007 10:14:50 +0000
From: Christian Praetorius 

Subject: Re: "PCR Methods and Applications"
To: methods from net.bio.net
Message-ID: <547t4iF1uufhtU3 from mid.individual.net>
Content-Type: text/plain; charset=us-ascii

wrote:

>hello, I'm very interesting by the book " PCR Methods and applications" but I don't kwon where it's possible tofind it, can you help me?

Enter the title of your book to amazon.com and you will find a lot of
books matching this. Take the ISBN and order via your local bookstore.

Christian

-- 
X-no-Sig: yes


------------------------------

Message: 9
Date: Fri, 23 Feb 2007 10:58:30 +0000
From: ChenHA 
Subject: Re: Pipetting, contamination....
To: methods from net.bio.net
Message-ID: <1172228314.29746.0 from proxy02.news.clara.net>
Content-Type: text/plain; charset=ISO-8859-1; format=flowed

DK wrote:
> In article <1172181256.088647.198980 from a75g2000cwd.googlegroups.com>, "peter" 
wrote:
>>> Jeez, I've done this experiment yesterday:
>>> 1. Took a 1 ml Pipetman, set it to 1 ml and pipetted overnight E.coli
>>> culture up and down 20 times.
>>> 2. Immediately after that, changed a tip, took a bottle of sterile
>>> Terrific Broth, pipetted 20 times, set at 37C for 20 hours.
>>>
>>> The result: no signs of bacterial growth today ==> no aerosol
>>> contamination.
>>>
>>> DK
>> Seems like a good experiment, I just wonder what volume of the TB
>> media you used?
> 
> The standard square bottle filled with 100 ml. I still have that bottle,
> so if you have your doubts, I can look at it very attentively by Monday. 
> But, you know, E.coli can grow anaerobically so shaking is not 
> strictly necessary...
> 
> I pretty much knew the outcome based on the fact that I am still 
> using the same ~1.5 years old bottle of SOC used for an umpteen 
> (more like ~ 100 :-)) 

You must be more careful than I am. I rarely reuse media bottles or 
flask once they have been opened. I know that they often stay clear for 
days after being opened, but still, I don't like taking chances.

I think a lot of this has to do with the environment (perhaps the air 
quality) you work in. I have worked in labs where I tried to be 
extremely careful, but still strange colourful colonies grew on plates 
after it's left on bench for some time. In other labs I rarely see this 
even though I was less careful.


> electroporations, each involving the same 
> 1 ml Pipetman and no filter tips all along. Amazingly, still no visible 
> growth in the bottle and, as recently as two days ago, 20 out of 20 
> clones analyzed contained the expected plasmid. 
> 
> DK
> 
> P.S. This topic reminded me of one curious observation: 
> 
> Everyone I know who does "microbiology" outside of sterile hood 
> is engaged in the practice of flaming necks of every bottle and 
> flask they use. When asked why they are doing it, the only answer 
> ever given is "to keep it sterile". Problem is, for as far as I can tell,
> the way it is done, it's a completely useless exercise in at least 
> 9 out of 10 times. 
> 

I don't flame anything, and don't understand why people insist on 
flaming. Flame in any case seems to cause more problems, for example, 
I have seen on more than one occasions where flame cause the plastic 
gloves people wear to go up in flame as well (spreader dipped in 
ethanol, flamed, flaming ethanol dripped onto gloves, whooomp!)


> 
> 


------------------------------

Message: 10
Date: Fri, 23 Feb 2007 11:08:43 +0000
From: ChenHA 
Subject: Re: Purifying PCR product from 2% agarose gel
To: methods from net.bio.net
Message-ID: <1172228928.20247.0 from damia.uk.clara.net>
Content-Type: text/plain; charset=ISO-8859-1; format=flowed

Ednot wrote:

> 
> On Feb 18, 10:46 am, "hilary cassidy" wrote:
>> Hi
>>
>> I've been trying to purify my PCR product from a 2% agarose gel all
>> week now as it needs to be purified prior to Topo cloning. I've used
>> both the QIAEX II Gel Purification kit by Qiagen and the Illustra DNA
>> and Gel Band Purification Kit by GE Healthcare. Each time I have
>> strong bands on the gel prior to cutting the bands, but everytime I
>> purify and run the DNA on a gel to check there is nothing!!!

I stopped using QIAEX because it doesn't work well all the time, 
especially for small fragments. I use Geneclean and it hasn't failed me 
yet. Geneclean has a special kit for very small fragment (Mermaid if I 
remember correctly), although trying this and the normal kits for ~200bp 
fragments doesn't seem to make much difference (both worked equally well).

How big is your fragment? If you fragment is very small, check that you 
haven't mistaken your oligos for PCR products.


------------------------------

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