7 replies to this topic

[hianghao]

I've done cloning from PCR products and wanted to purify the plasmid. My miniprep protocol requires me to incubate transformed E coli in LB broth for 12 hours before processing. I did. In the morning when i wanted to retrieve the overnight culture, i found that the incubater stopped shaking and there were a clump of whitish thing (E coli?) in all tubes. I've got no idea when did it stop.However, i took those samples and proceeded with miniprep. After miniprep, i ran a 1h 1% TBE gel (without treatment with RE 1st) for my plasmid and found no band. Was it due to no shaking during incubation? Or something went wrong in miniprep? Or i shouldn't run a gel for plasmid without treatment with RE? Is it a routine to use RE to check the plasmid before sending to sequence?

There is a thread in this forum where ppl mentioned that they didn't purify the plasmid after sequencing. If i dont do miniprep, in what medium should i transfer my single colony? Does it need extra treatment before sequencing?

When doing cloning, why should we put the bacteria in a incubater with shaking function? Was it to evenly distribute the bacteria all over the culture? So that they will have enough nutrients? Then why some protocols require higher speed and some, lower?

Edited by hianghao, 08 September 2010 - 12:17 AM.

[HomeBrew]

Shaking of the culture is done to insure adequate aeration.  A overnight static culture of E. coli should still yield plenty of cells for a miniprep -- you should have seen plasmid bands with or without restriction enzyme treatment.  You may be confusing sequencing with colony PCR -- you don't need to isolate plasmid DNA to see if your plasmid + insert is in any given colony -- you can do that by PCR with appropriate primers right from the colony.  You do, however, need to isolate plasmid DNA to sequence the plasmid or insert.

[Adrian K]

Did you include proper antibiotics when you incubate your E.coli? Yes, I agree with Homebrew, you can actually do a colony PCR on it to verify whether the insert is inside your E.coli before you proceed for miniprep.

Usually for an established protocol, a miniprep will be hardly goes wrong.

I suspect you had lost your recombinant strain and the clump might be something else.

Just my 2 cents.

Edited by adrian kohsf, 08 September 2010 - 03:26 AM.

[hianghao]

Will run new samples

Edited by hianghao, 08 September 2010 - 04:34 AM.

[hianghao]

HomeBrew, on 08 September 2010 - 03:15 AM, said:

Shaking of the culture is done to insure adequate aeration.  A overnight static culture of E. coli should still yield plenty of cells for a miniprep -- you should have seen plasmid bands with or without restriction enzyme treatment.  You may be confusing sequencing with colony PCR -- you don't need to isolate plasmid DNA to see if your plasmid + insert is in any given colony -- you can do that by PCR with appropriate primers right from the colony.  You do, however, need to isolate plasmid DNA to sequence the plasmid or insert.

I see... Thanks for the information!

adrian kohsf, on 08 September 2010 - 03:25 AM, said:

Did you include proper antibiotics when you incubate your E.coli? Yes, I agree with Homebrew, you can actually do a colony PCR on it to verify whether the insert is inside your E.coli before you proceed for miniprep.

Usually for an established protocol, a miniprep will be hardly goes wrong.

I suspect you had lost your recombinant strain and the clump might be something else.

Just my 2 cents.

Yes i did include antibiotics with the same concentration as my plate.
Do you mean that the recombinant strain is lost when transferring from plate to broth? OR it lost its function? What can i do for that?

I ran 2ul sample in gel. Tomorrow will try 5ul. Hopefully its because of low concentration..

[Adrian K]

hianghao, on 08 September 2010 - 04:34 AM, said:

adrian kohsf, on 08 September 2010 - 03:25 AM, said:

Did you include proper antibiotics when you incubate your E.coli? Yes, I agree with Homebrew, you can actually do a colony PCR on it to verify whether the insert is inside your E.coli before you proceed for miniprep.

Usually for an established protocol, a miniprep will be hardly goes wrong.

I suspect you had lost your recombinant strain and the clump might be something else.

Just my 2 cents.

Yes i did include antibiotics with the same concentration as my plate.
Do you mean that the recombinant strain is lost when transferring from plate to broth? OR it lost its function? What can i do for that?

I ran 2ul sample in gel. Tomorrow will try 5ul. Hopefully its because of low concentration..

Just to share: I remember the mistake I done few months ago with my E.coli JM107. I use my stock culture from -80C to perform cloning and I had selected the ampR strains which I thought is the recombinant strains, but when I prep it nothing comes out. I also thought might be the minprep kit malfunction initially. I use my stock culture to grow in my antibiotics plate (without any recombinant insert) I found that there are some colonies formed in my antibiotics plate, which means either my antibiotic plate was "expired" or there is contamination. I made a triple concentrated antibiotics plate and subculture the "survived" colony onto it and it still grow... this told me that actually my -80 stock had been contaminated with something. So, I had to buy a new host cells again... thats the reason myself and Homebrew suggest you to do a colony PCR to see if the insert was there before you proceed.

[hianghao]

adrian kohsf, on 08 September 2010 - 05:14 PM, said:

hianghao, on 08 September 2010 - 04:34 AM, said:

adrian kohsf, on 08 September 2010 - 03:25 AM, said:

Did you include proper antibiotics when you incubate your E.coli? Yes, I agree with Homebrew, you can actually do a colony PCR on it to verify whether the insert is inside your E.coli before you proceed for miniprep.

Usually for an established protocol, a miniprep will be hardly goes wrong.

I suspect you had lost your recombinant strain and the clump might be something else.

Just my 2 cents.

Yes i did include antibiotics with the same concentration as my plate.
Do you mean that the recombinant strain is lost when transferring from plate to broth? OR it lost its function? What can i do for that?

I ran 2ul sample in gel. Tomorrow will try 5ul. Hopefully its because of low concentration..

Just to share: I remember the mistake I done few months ago with my E.coli JM107. I use my stock culture from -80C to perform cloning and I had selected the ampR strains which I thought is the recombinant strains, but when I prep it nothing comes out. I also thought might be the minprep kit malfunction initially. I use my stock culture to grow in my antibiotics plate (without any recombinant insert) I found that there are some colonies formed in my antibiotics plate, which means either my antibiotic plate was "expired" or there is contamination. I made a triple concentrated antibiotics plate and subculture the "survived" colony onto it and it still grow... this told me that actually my -80 stock had been contaminated with something. So, I had to buy a new host cells again... thats the reason myself and Homebrew suggest you to do a colony PCR to see if the insert was there before you proceed.

I see... I'll check with my ampicillin 1st! Because i am using a kit that recommended 100ug/ml; but i was using 10ug/ml, following a protocol of my neighbour lab who use the same kit and it works for them.

Btwy, this is my gel. Miniprep at the right hand side of a 1kb marker. 7 lanes.

Edited by hianghao, 09 September 2010 - 12:32 AM.

[HomeBrew]

The amount of ampicillin to use is dictated by the level of resistance conferred by the marker on the plasmid you're using.  Be sure each liquid culture is begun with a pure isolate or a well-isolated single colony off an L-agar plate containing the appropriate amount of ampicillin.