7 replies to this topic

[sashay77]

Hi Guys!

I am trying to extract a 3 Mbp genome intact. Any Suggestions????

Thanks

Edited by sashay77, 10 September 2009 - 03:18 PM.

[swanny]

No first-hand experience, but would a protocol for extraction of yeast chromosomal DNA be appropriate, or something to modify to your purpose?

[phage434]

Look at protocols for pulse field gels. The basic technique is embedding the cells in an agarose gel, digesting the cell with proteinase-K, SDS, and then washing with TE, all in the form of a gel. DNA can be digested in this format as well. Finally, the agarose can be digested with beta-agarase to release the DNA. It will be extremely fragile, and not easily manipulated.

[sashay77]

Thanks Guys!

I am starting with PFGE using embedded cells in agarose. The most relevant protocol I found was http://aem.asm.org/c...full/71/11/6910 in which they were able to extract a 1.6 mbp DNA. Anyone gone higher than that???? Most of the other papers restrict digest their DNA before running on the gel.

@Phage434: Is it really necessary to digest the agarose? why not just put the just put the DNA embedded agarose into the well?
@Swanny: Could you please send me the protocol, or a paper. I could probably find some tips to prevent DNA shearing.

Appreciate the help guys!!!

Keep Discovering!!!!

Edited by sashay77, 11 September 2009 - 02:25 PM.

[sashay77]

Does it matter which agarose I use to make the plugs??? I used Nuseive and it seems to be breaking pretty easily and disappearing at 56C. Any recommendations???

Thanks!

[phage434]

There is essentially no chance to keep intact 3 Mbp genomic DNA intact in solution. Even with wide bore pipet tips, it will shear very easily. Typically, special agarose such as Incert agarose is used to prepare cell suspensions.

[sashay77]

Thanks Phage!

I am not using any pipettes, doing the complete lysis in the agarose and then directly transferring the agarose to Pulse field. I got a big smear. Is it even possible to get a 4.5 mbp genome into the gel? I am using 1% of Seakem Gold (esp recommended for LMV DNA). I am trying to tweak the protocol to see if that helps, but please feel free to suggest.

Sashay

[phage434]

I use a 2% Incert agarose in TE, heated then cooled to 50C, a washed cell suspension heated to 37C, mixed in equal proportion (final agarose concentration 1%). I cut the end off a 1 ml syringe and load the agarose in the syringe, and let it cool. I slice 1-2 mm slices of the agarose with a clean razor blade and resuspend in TE. The number of cells in the initial cell suspension is important, and you should prepare several versions with different dilutions of cells. You want a slightly milky look, but not too white.

Then a series of digestions and washes are done, first with high EDTA concentrations with proteinase-K and N-laurylsarcosine, heated to 50C for 12 hours, then a set of washes with TE. Washes are also at 50C. Following washes, the cells can be stored at 4C for quite a while in TE. I did the washes in a hybridization oven rotator in 50 ml centrifuge tubes.

Digestion is done by washing at least 2x in RE buffer, followed by RE buffer + RE at 37C, followed by TE washes at 50C.

The gel slices can then be loaded into gel slots of a 1% gel, and sealed with hot 1% agarose.

You probably are overloading he gel, or the problem might be that the DNA is still circular. You can cut most genomic DNA at the ribosomal RNA sites with the enzyme I-CeuI, which is helpful to linearize fragment and identify the number of rRNA genes.


I'll try to dig up a real protocol for you.
Have you gotten the high MW yeast DNA fragments to run well on your setup?
What timing/voltage/angle are you using?