Methods Digest, Vol 21, Issue 25

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.... ([Only registered users see links. ].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)


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Message: 1
Date: Fri, 23 Feb 2007 03:26:25 GMT
From: [Only registered users see links. ] (DK)
Subject: Re: Pipetting, contamination....
To: [Only registered users see links. ]
Message-ID:

In article <1172199429.419560.225640@p10g2000cwp.googlegroups .com>, "peter"
wrote:

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


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Message: 2
Date: Fri, 23 Feb 2007 05:58:29 GMT
From: [Only registered users see links. ] (DK)
Subject: Re: Pipetting, contamination....
To: [Only registered users see links. ]
Message-ID:

In article <1172202357.518158.176980@8g2000cwh.googlegroups.c om>, "peter"
wrote:


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





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

Subject: Re: Pipetting, contamination....
To: [Only registered users see links. ]
Message-ID: <1172199429.419560.225640@p10g2000cwp.googlegroups .com>
Content-Type: text/plain; charset="iso-8859-1"

On Feb 22, 9:09 pm, [Only registered users see links. ] (DK) wrote:
wrote:
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.



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

Subject: Re: Pipetting, contamination....
To: [Only registered users see links. ]
Message-ID: <1172202357.518158.176980@8g2000cwh.googlegroups.c om>
Content-Type: text/plain; charset="iso-8859-1"

On Feb 22, 10:26 pm, [Only registered users see links. ] (DK) wrote:
wrote:

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



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Message: 5
Date: Fri, 23 Feb 2007 09:54:46 +0000
From: "[Only registered users see links. ].uk"
Subject: Re: Pipetting, contamination....
To: [Only registered users see links. ]
Message-ID:

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

On Fri, 23 Feb 2007, DK wrote:
wrote:

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: [Only registered users see links. ]
Message-ID: <[Only registered users see links. ]>
Content-Type: text/plain; charset=us-ascii

[Only registered users see links. ] (DK) wrote:


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

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

Subject: Re: Pipetting, contamination....
To: [Only registered users see links. ]
Message-ID: <[Only registered users see links. ]>
Content-Type: text/plain; charset=us-ascii

[Only registered users see links. ] (DK) wrote:


If people work properly and clean.

Christian

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

Subject: Re: "PCR Methods and Applications"
To: [Only registered users see links. ]
Message-ID: <[Only registered users see links. ]>
Content-Type: text/plain; charset=us-ascii

wrote:


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


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Message: 9
Date: Fri, 23 Feb 2007 10:58:30 +0000
From: ChenHA
Subject: Re: Pipetting, contamination....
To: [Only registered users see links. ]
Message-ID: <[Only registered users see links. ].clara.net>
Content-Type: text/plain; charset=ISO-8859-1; format=flowed

DK wrote:
wrote:

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.



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: [Only registered users see links. ]
Message-ID: <[Only registered users see links. ].clara.net>
Content-Type: text/plain; charset=ISO-8859-1; format=flowed

Ednot wrote:


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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