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DNA extraction problems using alkaline lysis method - It stopped working! (Jun/21/2005 )

I am having some problems with extracting DNA from BACs (RP11 library) using the alkaline lysis method (which is eventually used for FISH). I would appreciate any ideas on what I could do.

I worked in this lab last year, and the method worked perfectly.
I picked a BAC out of the RP11 library, grew it up in a 3ml culture, plated it out to choose a single colony, then grew the single colony up in a 50ml falcon tube. From here, the plasmid DNA is extracted and isolated using the alkaline lysis method. The DNA is labelled with Dig-Nick to make a probe, and the probe is hybridised onto human chromosomes to check for deletions, or map breakpoints etc.

It worked as soon as I stepped into the lab (I was on a one year placement for university).

During the final few weeks, I was having some problems with getting a good probe signal, and when the new student came it the method pretty much stopped working all together (I had left, back to uni).

The problem has been narrowed down to the DNA extraction/isolation (so everything preceding this as well).

I would just like to know if anyone has any ideas on what could be going wrong.

They have tried:
- picking new BAC’s
- new Liquid Broth
- New solutions I, II and III
- New Dig-Nick, and nothing seemed to work.

Now I’m back for 2 weeks during uni holidays to see if I can have a hack at it. ph34r.gif

On day 3 I have noticed:
- when adding solution II to the resuspended cell pellet, it doesn’t seem as viscous or stringy (or gloopy, don’t really know how to describe it).
- When adding solution III, I used to get clumps of white precipitate, heaps of it! But now I think it looks different, its not as white and more stringy and fragile.

After taking the supernatant, incubating in isopropanol overnight and pelleting the DNA the next morn, the pellet is not a big translucent clump down the bottom, it’s a whiter/yellow color, and only translucent around the edges.

Tomorrow I will be adding Dig-nick and running a gel electrophoresis on a few BAC dna samples. I’ll post the results tomorrow.

I don’t really know what could be going wrong. Obviously it could be solution 2 not lysing cells to release DNA, which means that there is no junk for solution 3 to precipitate. But these solutions have been remade time and time again.
The method is so straight forward I can’t think of any stage tht can be stuffed up.
Maybe it could be the ratio of the solutions to cell number?? Or something with the cell growth or quality? I don’t know.


Goddammit that was an essay. I’m only in for 2 weeks until uni starts again, and I don’t want to waste 2 weeks worth of the lab’s money.

If you have any questions, please ask, and any ideas, please share wink.gif

Thanks,
James laugh.gif

-bizzarojames-

Hey
Are you sure you are using cold solution III and are incubating on ice after addition of solution II and solution III before centrifugation.

If yes then just try increasing the time of incubation
Good luck



QUOTE (bizzarojames @ Jun 21 2005, 11:56 PM)
I am having some problems with extracting DNA from BACs (RP11 library) using the alkaline lysis method (which is eventually used for FISH).  I would appreciate any ideas on what I could do.

I worked in this lab last year, and the method worked perfectly.
I picked a BAC out of the RP11 library, grew it up in a 3ml culture, plated it out to choose a single colony, then grew the single colony up in a 50ml falcon tube.  From here, the plasmid DNA is extracted and isolated using the alkaline lysis method.  The DNA is labelled with Dig-Nick to make a probe, and the probe is hybridised onto human chromosomes to check for deletions, or map breakpoints etc.

It worked as soon as I stepped into the lab (I was on a one year placement for university).

During the final few weeks, I was having some problems with getting a good probe signal, and when the new student came it the method pretty much stopped working all together (I had left, back to uni). 

The problem has been narrowed down to the DNA extraction/isolation (so everything preceding this as well).

I would just like to know if anyone has any ideas on what could be going wrong.

They have tried:
- picking new BAC’s
- new Liquid Broth
- New solutions I, II and III
- New Dig-Nick, and nothing seemed to work.

Now I’m back for 2 weeks during uni holidays to see if I can have a hack at it.  ph34r.gif

On day 3 I have noticed:
- when adding solution II to the resuspended cell pellet, it doesn’t seem as viscous or stringy (or gloopy, don’t really know how to describe it).
- When adding solution III, I used to get clumps of white precipitate, heaps of it!  But now I think it looks different, its not as white and more stringy and fragile.

After taking the supernatant, incubating in isopropanol overnight and pelleting the DNA the next morn, the pellet is not a big translucent clump down the bottom, it’s a whiter/yellow color, and only translucent around the edges. 

Tomorrow I will be adding Dig-nick and running a gel electrophoresis on a few BAC dna samples.  I’ll post the results tomorrow. 

I don’t really know what could be going wrong.  Obviously it could be solution 2 not lysing cells to release DNA, which means that there is no junk for solution 3 to precipitate.  But these solutions have been remade time and time again. 
The method is so straight forward I can’t think of any stage tht can be stuffed up.
Maybe it could be the ratio of the solutions to cell number?? Or something with the cell growth or quality? I don’t know.


Goddammit that was an essay. I’m only in for 2 weeks until uni starts again, and I don’t want to waste 2 weeks worth of the lab’s money. 

If you have any questions, please ask, and any ideas, please share wink.gif 

Thanks,
James  laugh.gif

-ramakn-

hi,

how is the new Sol II? I used to make this sol (freshly mix NaOH + SDS) just before adding to the cell suspension.

-dtle-

Ramakn - Yes, incubation is on ice for 5 minutes, and solution III is straight from the fridge.
I was going to try increasing the incubation time, but decided to try increasing the volume first (thought maybe there was insufficient solution 2 to lyse cells, therefore less dna). THinking back, that doesnt really make that much sense, however i made a duplicate 50ml culture and will try incubating for 10 mins.

Dtle - Yeah, i made each solution fresh and used the following day. Last year i would make up 500ml of each and only remake them when they ran out, which could be weeks or months, and they still worked smile.gif


Thank you both for the ideas. I have made some probe and they are running on a gel right now. Hopefully i get some good results.

Cheers,
james

-bizzarojames-