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#1 learner2010

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Posted 03 March 2010 - 02:01 PM

I wanna consult every master-hands here with a problem I had just met in previous days. I picked up my only one colony transformed bacterium DH5a on the Amp(+) LB-agar plate and to transfer it to Amp(+) LB liquid for to grow the cell at 37'C 255rpm overnight, but the DH5a didn't grows in the Amp(+) LB liquid in Falcon Tube, it still not grow after I change new LB liquid(pH7.4), so I suppose the concentration of the antibiotic was too high, so I do it again today as that to change the concentration from 9ul Amp.(100mg/ml) per 9ml LB liquid to 8ul Amp.(100mg/ml) per 9ml LB liquid, but I don't know whether is it right or not?
Thank you.

Edited by learner2010, 03 March 2010 - 02:04 PM.


#2 bob1

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Posted 03 March 2010 - 04:00 PM

Do I have this right?...

You did a transformation and plated it out and got only one colony at the end?

If so; there is something wrong with either your transformation, or the plasmid, or your plates. You should see many (greater than 50) colonies per transformation.

What controls for the transformation steps did you have?

#3 learner2010

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Posted 03 March 2010 - 04:46 PM

Hi, bob1,
I am sorry my English is not good, I am a chinese. I mean that I got many colonies but I only pick it one colony up & transfer to in the LB-Amp liquid for cell growth. other one, I don't have any controls for the transformation steps, so what kinds of sample can be take as a control? Thank you,


Do I have this right?...

You did a transformation and plated it out and got only one colony at the end?

If so; there is something wrong with either your transformation, or the plasmid, or your plates. You should see many (greater than 50) colonies per transformation.

What controls for the transformation steps did you have?



#4 bob1

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Posted 04 March 2010 - 04:17 PM

Hi,

You usually need to screen a few (10-20) colonies to get a clone with the correct sequence. Occasionally one of the clones will not have the insert or will lose it, but still grow on the plates. Usually these will not grow when picked and placed into liquid medium. Sometimes clones will take a couple of days in liquid medium to grow to a suitable level from a picked colony.

The controls you should have used in your transformation are:
1) off target plasmid (e.g. pUC 18) to check that your transformation worked. Should be lots of colonies on this plate too.
2) unligated mix (your linearised plasmid without the insert) to check that the colonies are likely to be the result of a circular DNA and that your digestion worked properly. Should be very few colonies on this plate.
3) untransformed bacteria - to check that your antibiotics are good - you should get only 1 or 2 colonies on this plate at the most, usually you should get none.

#5 learner2010

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Posted 09 March 2010 - 09:55 AM

Hi,
Thank you for your suggest, it is important to a biginner, I will do it at next test. I believed my T4 DNA ligase was not work after I analysed all of results of late experiment, so I will begin to do new ligatioin reaction when I get a new T4 DNA ligase.
Lijun.

Hi,

You usually need to screen a few (10-20) colonies to get a clone with the correct sequence. Occasionally one of the clones will not have the insert or will lose it, but still grow on the plates. Usually these will not grow when picked and placed into liquid medium. Sometimes clones will take a couple of days in liquid medium to grow to a suitable level from a picked colony.

The controls you should have used in your transformation are:
1) off target plasmid (e.g. pUC 18) to check that your transformation worked. Should be lots of colonies on this plate too.
2) unligated mix (your linearised plasmid without the insert) to check that the colonies are likely to be the result of a circular DNA and that your digestion worked properly. Should be very few colonies on this plate.
3) untransformed bacteria - to check that your antibiotics are good - you should get only 1 or 2 colonies on this plate at the most, usually you should get none.






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