Re: [ccp4bb] AW: [ccp4bb] hi / cloning stuff

[Artem Evdokimov]

Hi,

While I do not think that the comments (at least those posted to the board)
were evil or derogatory, I nevertheless would like to emphasize the need for
details. 

With appropriate cloning strategy artefacts such as those described by Jan
could be avoided almost entirely. While 'in silico' cloning may be helpful,
consideration of major factors that can affect the process is much more
important. In silico stuff can be a bit misleading - for an example I highly
recommend interested parties to check out Anton Yuryev's book 'PCR primer
design'; specifically chapter 3 which discusses several common myths
regarding PCR primer design (it turns out that casually designed PCR primers
only work in ~70-75% of cases). Casual use of programs does not make things
better.

Overall, there are too many unknown branch points in a 'straightforward'
process such as cloning and without details the diagnosis of a problem
becomes equivalent to leaving your car mechanic a phone message 'my car does
not start, tell me how I can fix it'. Undoubtedly the mechanic would want to
know a little more about the problem at hand before suggesting an answer. It
is didactic to read about cases like this one, and this list is being read
by many - including both molecular biology professionals as well as novices.
So, details are useful and not just to the person who asked the question.

For example - if no colonies form after ligation, assuming that all the
basics have been checked (such as, properly designed amplification primer
sequences, compatible sticky ends, compatible enzymes, & suitable
restriction buffers to name just a few) the typical causes that should be
investigated further are:

0. Bad/incorrect/old/warmed-up competent cells. Imagine a freezer dying
briefly in the night, then recovering without anyone knowing. If you're
lucky your freezers are alarmed and chart-recorded. Yet even this still does
not prevent another user from carelessly taking competent cells out for too
long, not noticing, or failing to replace the compromised stock.
1. Too much insert - resulting in 1:2 ligation with incompatible sticky
ends.
2. Toxic insert - which is why the e.g. T7 promoter is oh so good since
cloning-grade cells do not have the right polymerase for it.
3. Star activity or unexpected cleavage site (i.e. the genetic database
tells you one thing but your source DNA is from a mutant).
4. Old/bad enzymes (almost does not happen these days although some brands
of DNA ligase expire faster than others).
5. Contaminated enzymes (happens more than we'd all like to see in labs with
multiple users of the same reagents).
6. Wrong/contaminated antibiotics (see previous comment regarding multiple
users)
7. mis-synthesized primers. Just a couple of weeks ago we had this happen -
a desperate sequencing attempt (of a third-step intermediate) confirmed an
error in one of the primers we used to produce a complicated construct that
kept failing time and time again.

And that's just a few options, and only for one specific case - 'no colonies
after ligation, transformation, and plating'. 

On a much more positive note, the overall process works. It has been
developed over decades, with continuous improvement by many extremely
talented people. But don't let the apparent simplicity fool you - there are
pitfalls present at every level. Still, it works more times than it does not
- and it's a whole lot easier than it used to be (cesium chloride gradients,
anyone?). We just had an intern (undergraduate student) who came in with
basic lab skills and had no prior experience in molecular biology,
crystallography, etc. but was able to clone, express, purify, and
crystallize a novel (soluble bacterial) protein. We solved the structure
before his internship ended. So, overall the process clearly works :)

Artem

-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Sridhar Prasad
Sent: Sunday, August 31, 2008 12:28 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] AW: [ccp4bb] hi / cloning stuff

I want to draw attention to some of the early unruly response(s) to this
question. 

First of all, no one is forcing anyone to respond to each and every message
that is posted on the CCP4 bulletin board. 

If you are so keen on responding, please be respectful and considerate and
think twice before you click on the send icon on your computer with
derogatory remarks.


Thanks
Sridhar Prasad

-Original Message-
From: CCP4 bulletin board on behalf of Jan Schoepe
Sent: Sat 8/30/2008 8:52 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] AW: [ccp4bb] hi / cloning stuff
 
Hi, I think your question is quite reasonable no matter what others say
about it. There were even people asking about how to make buffers and then a
big discussion about the HH equation arose. And we should not forget that
modern crystallization starts with cloning ;-) Anyway, you sho

Re: [ccp4bb] AW: [ccp4bb] hi / cloning stuff

[Dima Klenchin]

I found out that my glycerol stocks were not alright any more and I had no 
plasmid backups (do it!!!). So I had to clone, again.


Hint for the future: Dead glycerol stocks, even dead (say, a year old) 
clone on a plate, still contain your plasmid. Do a regular miniprep with 
it, transform - 99% of clones will contain your plasmid.


Dima






But in this case I used a cloning kit from Qiagen (I have no commercial 
interests here). I got the insert into the cloning vector from Qiagen very 
easily. Then I was able to amplify it as much as I wanted and the ligation 
procedure was not so dependent from the yield of the PCR reaction (high 
molar access of insert compared to the cleaved vector) . Also in this 
case, cloning into the expresson vector was not completely trivial (maybe 
1-1.5 months) but much easier than without this kit. So, it might be an 
idea to use a kit that you can amplify your insert as much as you want. I 
know that these kits a pricy but your time is also expensive and you also 
use/waste a lot of chemicals for unsuccessful trials.
After I finished my thesis, another PhD student started to work with these 
vectors and tried it for more than one year and then gave it up! So this 
vector really seems to be a tough one. Thats why if nothing works, you 
might take another vector into consideration. But before that you should 
check buffers, ligase, restriction enzymes etc. and do in silico cloning. 
Another idea would be do to de/phosphorylation (see textbooks).


Good luck!
Jan

--- vijay srivastava <[EMAIL PROTECTED]> schrieb am Sa, 30.8.2008:
Von: vijay srivastava <[EMAIL PROTECTED]>
Betreff: [ccp4bb] hi
An: CCP4BB@JISCMAIL.AC.UK
Datum: Samstag, 30. August 2008, 13:04

hi
i am facing problem in cloning,getting my insert and vector at the
correct position after digestion but after ligation colony is not
coming and if how it is coming than i am not getting my insert



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Re: [ccp4bb] AW: [ccp4bb] hi / cloning stuff

[Sridhar Prasad]

I want to draw attention to some of the early unruly response(s) to this 
question. 

First of all, no one is forcing anyone to respond to each and every message 
that is posted on the CCP4 bulletin board. 

If you are so keen on responding, please be respectful and considerate and 
think twice before you click on the send icon on your computer with derogatory 
remarks.


Thanks
Sridhar Prasad

-Original Message-
From: CCP4 bulletin board on behalf of Jan Schoepe
Sent: Sat 8/30/2008 8:52 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] AW: [ccp4bb] hi / cloning stuff
 
Hi, I think your question is quite reasonable no matter what others say about 
it. There were even people asking about how to make buffers and then a big 
discussion about the HH equation arose. And we should not forget that modern 
crystallization starts with cloning ;-) Anyway, you should have given us some 
more details about your experiments (cells, vectors etc.) and maybe a better 
subject.
If you want to clone "something", it is a good idea to do it in silico (e.g. 
with Clone Manager or Vector NTI) before you walk into a wet lab to check, that 
things can work as desired or if there are fishy things like internal 
restriction sides etc. 
Let me tell you a story from my PhD thesis, maybe this will help you somehow. I 
started with cloning 10 genes into a before modified pET vector (for E. coli 
overexpression). After cleavage of the vector I got cohesive ends ("sticky 
ends"). The genes were PCR amplified  from the chromosome of another bacterium. 
So things should have been very straight forward. Sticky ends should make sure 
that the amplified genes were inserted in the right direction and the cleaved 
vector should not ligate without the insert (and of course there was antibiotic 
selection). But what I observed was often quite different from that. Often agar 
plates stayed empty after transformation. That means, the cells were not able 
to grow, because there was no vector in the cells that made them be able to 
survive on the antibiotic plates. When I got colonies, the DNA gel looked very 
strange (not possible to interpret) or the vector closed without the insert. In 
this case, E. coli seems
 to be able to "repair" the cohesive ends. Finally, I got it done but it took 
me months. In one case, a plate seemed to be empty but I did not wanted to 
throw it away so I let it stay a litte longer (~another day?, I do not remeber 
exactly, but it was for a long time...) at 37° C than just over night (~0.5-1 
days). Finally, one single colony showed up and it was one of the right ones. 
With another vector it might be possible to do the same thing within weeks. 
This is just cloning luck.
Another story, even if it could be embarassing to mention it here. In one case, 
I got an Xtal structure but then I wanted to do some more activity tests. I 
found out that my glycerol stocks were not alright any more and I had no 
plasmid backups (do it!!!). So I had to clone, again. But in this case I used a 
cloning kit from Qiagen (I have no commercial interests here). I got the insert 
into the cloning vector from Qiagen very easily. Then I was able to amplify it 
as much as I wanted and the ligation procedure was not so dependent from the 
yield of the PCR reaction (high molar access of insert compared to the cleaved 
vector) . Also in this case, cloning into the expresson vector was not 
completely trivial (maybe 1-1.5 months) but much easier than without this kit. 
So, it might be an idea to use a kit that you can amplify your insert as much 
as you want. I know that these kits a pricy but your time is also expensive and 
you also use/waste a lot of
 chemicals for unsuccessful trials.
After I finished my thesis, another PhD student started to work with these 
vectors and tried it for more than one year and then gave it up! So this vector 
really seems to be a tough one. Thats why if nothing works, you might take 
another vector into consideration. But before that you should check buffers, 
ligase, restriction enzymes etc. and do in silico cloning. Another idea would 
be do to de/phosphorylation (see textbooks).

Good luck!
Jan

--- vijay srivastava <[EMAIL PROTECTED]> schrieb am Sa, 30.8.2008:
Von: vijay srivastava <[EMAIL PROTECTED]>
Betreff: [ccp4bb] hi
An: CCP4BB@JISCMAIL.AC.UK
Datum: Samstag, 30. August 2008, 13:04


hi
i am facing problem in cloning,getting my insert and vector at the 
correct position after digestion but after ligation colony is not 
coming and if how it is coming than i am not getting my insert
 




   Unlimited freedom, unlimited storage. Get it now

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Massenmails. 
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[ccp4bb] AW: [ccp4bb] hi / cloning stuff

[Jan Schoepe]

Hi, I think your question is quite reasonable no matter what others say about 
it. There were even people asking about how to make buffers and then a big 
discussion about the HH equation arose. And we should not forget that modern 
crystallization starts with cloning ;-) Anyway, you should have given us some 
more details about your experiments (cells, vectors etc.) and maybe a better 
subject.
If you want to clone "something", it is a good idea to do it in silico (e.g. 
with Clone Manager or Vector NTI) before you walk into a wet lab to check, that 
things can work as desired or if there are fishy things like internal 
restriction sides etc. 
Let me tell you a story from my PhD thesis, maybe this will help you somehow. I 
started with cloning 10 genes into a before modified pET vector (for E. coli 
overexpression). After cleavage of the vector I got cohesive ends ("sticky 
ends"). The genes were PCR amplified  from the chromosome of another bacterium. 
So things should have been very straight forward. Sticky ends should make sure 
that the amplified genes were inserted in the right direction and the cleaved 
vector should not ligate without the insert (and of course there was antibiotic 
selection). But what I observed was often quite different from that. Often agar 
plates stayed empty after transformation. That means, the cells were not able 
to grow, because there was no vector in the cells that made them be able to 
survive on the antibiotic plates. When I got colonies, the DNA gel looked very 
strange (not possible to interpret) or the vector closed without the insert. In 
this case, E. coli seems
 to be able to "repair" the cohesive ends. Finally, I got it done but it took 
me months. In one case, a plate seemed to be empty but I did not wanted to 
throw it away so I let it stay a litte longer (~another day?, I do not remeber 
exactly, but it was for a long time...) at 37° C than just over night (~0.5-1 
days). Finally, one single colony showed up and it was one of the right ones. 
With another vector it might be possible to do the same thing within weeks. 
This is just cloning luck.
Another story, even if it could be embarassing to mention it here. In one case, 
I got an Xtal structure but then I wanted to do some more activity tests. I 
found out that my glycerol stocks were not alright any more and I had no 
plasmid backups (do it!!!). So I had to clone, again. But in this case I used a 
cloning kit from Qiagen (I have no commercial interests here). I got the insert 
into the cloning vector from Qiagen very easily. Then I was able to amplify it 
as much as I wanted and the ligation procedure was not so dependent from the 
yield of the PCR reaction (high molar access of insert compared to the cleaved 
vector) . Also in this case, cloning into the expresson vector was not 
completely trivial (maybe 1-1.5 months) but much easier than without this kit. 
So, it might be an idea to use a kit that you can amplify your insert as much 
as you want. I know that these kits a pricy but your time is also expensive and 
you also use/waste a lot of
 chemicals for unsuccessful trials.    
After I finished my thesis, another PhD student started to work with these 
vectors and tried it for more than one year and then gave it up! So this vector 
really seems to be a tough one. Thats why if nothing works, you might take 
another vector into consideration. But before that you should check buffers, 
ligase, restriction enzymes etc. and do in silico cloning. Another idea would 
be do to de/phosphorylation (see textbooks).

Good luck!
Jan

--- vijay srivastava <[EMAIL PROTECTED]> schrieb am Sa, 30.8.2008:
Von: vijay srivastava <[EMAIL PROTECTED]>
Betreff: [ccp4bb] hi
An: CCP4BB@JISCMAIL.AC.UK
Datum: Samstag, 30. August 2008, 13:04


hi
i am facing problem in cloning,getting my insert and vector at the 
correct position after digestion but after ligation colony is not 
coming and if how it is coming than i am not getting my insert
 




   Unlimited freedom, unlimited storage. Get it now

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Do You Yahoo!?
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