cloning template

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cloning template

Postby creepster » Jan 04 2007 4:32 pm

it seems that the majority of people include too little information about their cloning experiment, and therefore trouble shooting fails until they provide further information
to avoid this (maybe) i tried to come up with a template, so you can fill in the gaps or X the method/way chosen whenever necessary

please feel free to edit or even add steps i have forgotten, so we might come up with a nice form to fill out
it looks much better in word, when each new bulletin sublevel is pushed inwards

cheers


1. preparation of vector:
1.1. vector: _________
1.2. vector resistance: _________
1.3. vector size: _______ kb
1.4. vector was:
1.4.1. ___ double digested with ____ units or ___ ul of REN _____ and untis or ___ ul of REN _____ in ______ buffer
1.4.2. ___ sequential digested in a total volume of ____ ul first with ____ units or ___ ul of REN _____ and then with untis or ___ ul of REN _____ adding ____ buffer
1.4.2.1. vector was purified by ___ column; ___ agarose gel
1.4.3. ___ dephosphorylated; ___ not dephosphorylated
1.4.3.1. vector was purified by ___ column; ___ agarose gel

2. preparation of insert:
2.1. insert size: _______ bp
2.2. insert was:
2.2.1. ___ PCR amplified with primers including REN sites _____ and _____
2.2.1.1. insert was purified after PCR by ___ column; ___ agarose
2.2.2. cut out of another vector by REN ___ double digest; ___ sequential digest in a total volume of ____ ul with ____ units or ___ ul of REN _____ and untis or ___ ul of REN _____
2.2.2.1. insert was purified after digest by ___ column; ___ agarose
2.2.3. ___ PCR amplified and treated to create blunt ends by _____; subsequent digested by REN _____ to create one sticky end
2.2.3.1. insert was purified after digest by ___ column; ___ agarose

3. ligation set up:
3.1. ratio of vector : insert is ___ : ___
3.2. concentration of vector : insert is ____ ng : ____ ng
3.3. ligation was done in ___ ul total volume @ ___ ºC for ___ o/n; ___ hrs

4. transformation:
4.1. transformation is done by ___ calciumchloride/heat shock; ___ electroporation with bacteria strain _________
4.2. transformation set up included following controls:
4.2.1. ___ positive control; a known unaltered plasmid (expect a lot of colonies)
4.2.2. ___ negative control; linearized vector submitted to ligation
4.2.2.1. ___ dephosphorylated (expect no colonies)
4.2.2.2. ___ not dephosphorylated
4.2.2.2.1. ___ expect lots of colonies if both ends are blunt, same REN site or have same overhangs
4.2.2.2.2. ___ expect no/few colonies if both ends have different REN sites
4.3. ___ bacteria were regenerated @ 37ºC in _______ media
4.4. bacteria were streaked out on _______ plates

5. results:
5.1. ______ colonies for positive control
5.2. ______ colonies for negative control
5.3. ______ colonies for cloned vector
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Postby r.rosati » Jan 04 2007 7:08 pm

Thanks Creepster, great job! I made the thread sticky!

-Rob
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Postby niches » Jan 07 2007 5:00 am

what does this mean?
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Postby r.rosati » Jan 07 2007 1:45 pm

Well, basically it means that when someone posts something like "Hello, i tried to clone a PCR product and got no colonies, why?" we can just answer "please look at (this post) and provide the informations listed there, or it'll be hard to tell what went wrong".

Until now, most of the times people had to ask "ok but how many micrograms? What is your vector? How long is your insert?" and so on, which gets a bit annoying when you have to do it for the 20th time...

-Rob
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Re: cloning template

Postby mchlbrmn » Jan 07 2007 7:27 pm

I attempted to put indents in for you using the image or list or list= buttons, but couldn't do it. I was able to do this in the past, but can't figure it out today. Instead I added "`"s in place of blank indents.
I proof read and revised the form attempting to clarify. It may be a bit cumbersome, I'm not sure if I made it better or worse. Please feel free to use this, edit further, or revert to the old form as people think is best.


Cloning Trouble Shooting Information form.
Please "X" or put information in the underlines, and modify the form as makes sense for your experiment.
RE = restriction enzyme.
(If you indicate ul of RE, we will assume the common concentration of 10-20 u/ul.)

1. preparation of vector:
`1.1. vector: _________
``1.2. vector resistance: _________
``1.3. vector size: _______ kb
``1.4. vector was:
```1.4.1. ___ Double digested with ____ units or ___ ul of REN ___ and__ units or ___ ul of REN _____ in ___ul buffer.
```1.4.2. ___ Sequentially digested first with ____ units or ___ ul of REN ___ in __ ul volume, and then adding __units or __ ul of REN ____adding ___ul buffer ___.
```1.4.2.1. vector was purified by ___ column; __ agarose gel; or __ precipitation.
```1.4.3. ___ dephosphorylated; ___ not dephosphorylated ; __ end filled.
```1.4.3.1. vector was purified by ___ column; ___ agarose gel; __ precipitaton.

2. preparation of insert:
`2.1. insert size: __ bp
``2.2. insert was:
```2.2.1. ___ PCR amplified with primers including REN sites _____ and ___ by the polymerase (PCR enzyme) ____ in __ volume reaction.
````2.2.1.1. Insert was purified after PCR by ___ column; ___ agarose.
```2.2.2. ___ PCR product treated to create blunt ends by enzyme ____; subsequently (digested by REN _____ to create one sticky end).
````2.2.2.1. insert was purified by ___ column; ___ agarose; __ precipitation.
```2.2.3. cut out of another vector by REN(s) ___ ( check here___ if sequential digest) in a total volume of ____ ul with __ units or __ ul of REN ____ and __ units or __ ul of REN ____.
````2.2.3.1. insert was purified after digest by ___ column; ___ agarose; __ precipitation.

3. ligation set up:
`3.1. ratio of vector : insert is ___ : ___
`3.2. concentration of vector : insert is ____ ng : ____ ng in __ volume.
`3.3. ligation was done @ ___ ºC for ___ o/n; ___ hrs with T4 ligase.

4. transformation:
`4.1. transformation is done by ___ calciumchloride/heat shock; ___ electroporation with __ ul bacteria strain ______.
`4.2. transformation set up included following controls:
``4.2.1. ___ positive control; a known unaltered plasmid (expect a lot of colonies)
``4.2.2. ___ negative control; linearized vector submitted to ligation
```4.2.2.1. ___ dephosphorylated vector. (expect no colonies)
```4.2.2.2. ___ not dephosphorylated vector.
````4.2.2.2.1. expect lots of colonies if both ends are blunt, same REN site or have same overhangs
````4.2.2.2.2. expect no/few colonies if both ends have different REN sites
`4.3. Bacteria were regenerated @ 37ºC in ____ul media
`4.4. __ ul bacteria were streaked out on ____ plates

5. results:
`5.1. ______ colonies for positive control
`5.2. ______ colonies for negative control
`5.3. ______ colonies for new/experimental cloning
``5.3.1 __Clones were obtained but had following problems:____[/quote]


In 4.2.2.2.1 and ....2 there was a "__". Was information expected to be inputted? I deleted them.
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Re: cloning template

Postby creepster » Jan 07 2007 9:07 pm

mchlbrmn wrote:In 4.2.2.2.1 and ....2 there was a "__". Was information expected to be inputted? I deleted them.


awsome editing, i didnt think of using the ' to make tabs

4.2.2.2.1 and ...2 were ment to be "X"ed, depending on what the cloner thought to the previous preparation and ligation
so to say an interna control if they thought correct
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Re: cloning template

Postby r.rosati » Jan 07 2007 9:58 pm

Indents can be inserted within a "code" format, like this:



Cloning Trouble Shooting Information form.
Please "X" or put information in the underlines, and modify the form as makes sense for your experiment.
RE = restriction enzyme.
(If you indicate ul of RE, we will assume the common concentration of 10-20 u/ul.)

Code: Select all
1. preparation of vector:
1.1. vector: _________
1.2. vector resistance: _________
1.3. vector size: _______ kb
1.4. vector was:
  1.4.1. ___ Double digested with ____ units or ___ ul  of REN ___ and__ units or ___ ul of REN _____ in  ___ul  buffer.
  1.4.2. ___ Sequentially digested first with ____ units or ___ ul  of REN ___ in __ ul volume, and then adding __units or __ ul of REN ____adding ___ul buffer ___.
  1.4.2.1. vector was purified by ___ column; __ agarose gel; or __ precipitation.
  1.4.3. ___ dephosphorylated; ___ not dephosphorylated ; __ end filled.
   1.4.3.1. vector was purified by ___ column; ___ agarose gel; __ precipitaton.

2. preparation of insert:
2.1. insert size: __ bp
2.2. insert was:
  2.2.1. ___ PCR amplified with primers including REN sites _____ and ___ by the polymerase (PCR enzyme) ____ in __ volume reaction.
   2.2.1.1. Insert was purified after PCR by ___ column; ___ agarose.
  2.2.2. ___ PCR product treated to create blunt ends by enzyme ____; subsequently (digested by REN _____ to create one sticky end).
   2.2.2.1. insert was purified by ___ column; ___ agarose; __ precipitation.
  2.2.3. cut out of another vector by REN(s) ___  ( check here___  if sequential digest) in a total volume of ____ ul with __ units or __ ul  of REN ____ and __ units or __ ul of REN ____.
   2.2.3.1. insert was purified after digest by ___ column; ___ agarose; __ precipitation.

3. ligation set up:
3.1. ratio of vector : insert is ___ : ___
3.2. concentration of vector : insert is ____ ng : ____ ng in __ volume.
3.3. ligation was done @ ___ ºC for ___ o/n; ___ hrs with T4 ligase.

4. transformation:
4.1. transformation is done by ___ calciumchloride/heat shock; ___ electroporation with __ ul bacteria strain ______.
4.2. transformation set up included following controls:
  4.2.1. ___ positive control; a known unaltered plasmid (expect a lot of colonies)
  4.2.2. ___ negative control; linearized vector submitted to ligation
   4.2.2.1. ___ dephosphorylated vector. (expect no colonies)
   4.2.2.2. ___ not dephosphorylated vector.
    4.2.2.2.1.  ___ expect lots of colonies if both ends are blunt, same REN site or have same overhangs
    4.2.2.2.2.  ___ expect no/few colonies if both ends have different REN sites
4.3. Bacteria were regenerated @ 37ºC in ____ul media
4.4. __ ul bacteria were streaked out on ____ plates

5. results:
5.1. ______ colonies for positive control
5.2. ______ colonies for negative control
5.3. ______ colonies for new/experimental cloning
  5.3.1  __Clones were obtained but had following problems:____
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Postby r.rosati » Jan 07 2007 10:45 pm

An alternative to this form would be to present a "standard" post, like this one:

I am trying to clone a (insert type) into (vector).
The vector is
(standard or modified? If modified, containing what?).
(You may post a link containing basic informations on the vector; otherwise:)
The vector is (x) kb long and confers (antibiotics) resistance.
I'm preparing the vector for ligation in this way:
(please include all digestions, enzymatic treatments and purification steps. Feel free to post details if you don't feel sure about something, e.g. if you're using the right amount of RE or phosphatase).
I checked my prepared vector on gel (before ligation, after digestions) and I saw (correct length? no spurious bands?)
My insert is (x) bp long and...
(please include any informations you think appropriate, like high GC content, presence of repetitive sequences, or known "difficult'" region).
I prepare my insert for ligation this way: (again, please include all informations about enzymatic treatments or purifications. If you're digesting a PCR product, please note that many restriction enzymes fail to cut at sites near the ends of dsDNA)
I checked my insert on gel before ligation (if it makes sense) and I saw (correct length?)
I am ligating (x) ng of vector with (x) ng of insert in a (x) ul reaction, at (temperature) for (time).

(Could you tell if the ligation reaction actually produced the desired plasmid? If yes, how?)
I am transforming (commercial, home-made?) (strain) cells by (method). I know these cells are OK because (...)

I am getting the following results:
My ligation yields (approx number) of colonies wich by my standards are
(a fair amount, too few, an uncommonly humongous quantity)
I check my clones by (RE digestion? Sequencing?) and the problem is that (explain the problem as you can)

To troubleshoot things myself, I performed the following controls in parallel with my ligation (that is, together with your ligation, with the same batch of cells):
I tried to transform with (ng) of undigested vector and got (the usual enormous quantity of colonies, fewer colonies than usual)
I transformed with (ng) of unligated, prepared vector and got (x) colonies which by my standards are (a fair result, an unusually high amount of clones?)
I also transformed with a ligation containing only (ng) of vector and no insert, and I got (x) colonies which I think are (quite OK, too many, less than I expected)

I also tried to (sequence some clones? Change conditions, how?) but the results were that (explain?).
Last edited by r.rosati on Jan 08 2007 2:34 pm, edited 2 times in total.
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Postby creepster » Jan 07 2007 10:48 pm

hehehe

this is cool, there's probably forms like that possible for almost everything, like pouring SDS-PAGE, western transfer and antibody probing, immunostaining, real time PCR, etc, etc

the only challenge will be to get people to actually use it, i am counting the hours until there's another "i tried cloning my gene but it seems to not work, NE1 cn help plz ?!?!?!?!?!?!?! :?: :!: :shock: :roll: "


k, thx, bye
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Postby r.rosati » Jan 07 2007 10:56 pm

creepster wrote:i am counting the hours until there's another "i tried cloning my gene but it seems to not work, NE1 cn help plz ?!?!?!?!?!?!?! :?: :!: :shock: :roll: "


Agree :wink:
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Postby mchlbrmn » Jan 08 2007 3:12 am

I thought I tried the "code" button, but it didn't work for me.

Weird, the "standard" post looks good. I suggest soliciting whether the insert is a PCR product or cut from cloned DNA more directly.

I was going to say that in the "standard post" it was good that it was all bold or italics because the responder's reply can be easily distinguished if they insert it. ...However, if I press the "quote" to do this, it looks rather confusing with all the italics and bold indicators sprinkled throughout interupting it.

Perhaps both templates could be put in place and the user could decide which to use.
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Re: cloning template

Postby LifeTein » May 13 2009 1:07 am

You guys really do nice work.
James Chou, Ph.D.
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Re: cloning template

Postby abigen » Sep 27 2010 1:41 pm

thought I tried the "code" button, but it didn't work for me.

Weird, the "standard" post looks good. I suggest soliciting whether the insert is a PCR product or cut from cloned DNA more directly.

I was going to say that in the "standard post" it was good that it was all bold or italics because the responder's reply can be easily distinguished if they insert it. ...However, if I press the "quote" to do this, it looks rather confusing with all the italics and bold indicators sprinkled throughout interupting it.

Perhaps both templates could be put in place and the user could decide which to use.


www.abigen.com info@abigen.com
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Re: cloning template

Postby violahearz » Aug 31 2013 9:05 am

I am still using this template for my work...
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