concentration of my starting material?

[seqgirl123]

I am a little confused as I see two different concentrations required to start sample prep of genomic DNA for solexa.

One states to start with genomic dna that is 100ng-1ug in concentration to shear (found here, page 63). The other (here, page 7) says to start with 1ug-5ug of purified DNA to shear.

My question is what is the starting concentration to use upon shearing the DNA?

My other question is, when you go to the end-repair step, won't the starting concentration be diluted down? So if I start with 1ug of genomic DNA, when I perform the end repair step, it will be 300ng (30ul/100ul * 1ug = 0.3ug) DNA concentration after end repair. Or if I start with 100ng of genomic DNA, it will be 30ng DNA concentation after end-repair. I am taking the enzymes into account here of course with the dilution. Even if I dilute with the water only (45ul), it would be 667ng or 66 ng respectively. Does it not matter what starting concentration I use for shearing because it will be diluted downstream anyway?

[huguesparri]

Hello,
I'm not sure I understand your second question.
All I can say is that after the shearing, you purify ALL your sheared DNA using a Qiaquick column, ending with 30 µl of 5 µg (if you started with 5 µg) sheared DNA.
Then you use ALL of this eluate to perform the end repair.
After the Qiaquick purification, you should end up with 30 µl of 5 µg end repaired sheared DNA.

The only time when you don't use all of your sample is the PCR step where you only amplify 1 µl out of your 30 µl adapter modified DNA fragments.

[ScottC]

Yes, that's right. At each step you purify your modified DNA on a column and elute in a volume suitable for the next procedure. You won't recover it all of course, because the yield from these columns (or any other method) is never 100%. So if you start with 5ug, you won't actually end up with 5ug after each step. You will also lose a lot of your DNA at the size-selection step when you cut it from a gel; a great deal will not be in the size range you want and therefore it'll be left behind. However, it really doesn't matter, because the quantity that is loaded onto a lane of the flowcell is very small.

When it comes down to it, the concentration of DNA really doesn't matter, provided that you start with enough so that the losses that happen in each step don't result in your sample being so dilute at the end that there's nothing left.

In reality, we generally deal with 1-5ug, simply because it's usually available at that level (bacterial genomic DNA)... but I know you can get away with a lot less.

[mitchelS]

molar ratio of adaptor to DNA

Hi Scott,

I'm hoping you can help!

I have just sonicated my 1ug of DNA using the Bioeruptor so in theory I shouldn't have lost any material.

My question is about the ligation of the adaptor oligo mix following end-repair and A-tail. Illumina recommends using a 10:1 molar ratio of adaptor to DNA. How would you go about working out this considering I'm starting with 1ug and not 5ug?

Thanks for your help!

Cheers,

Mitch.

[sandyott]

bioruptor

Hey,

So what do you think of the bioeruptor??

have you ever tried covaris? we had a covaris Demo, just still tyring to get the funding to get one in here, but bioruptor seems soo much cheaper...

[ScottC]

Hi Mitch,

I don't normally worry too much about it. As long as you have enough adaptor present (i.e. the amount you'd use for 5ug), then it'll be fine. I have lowered the amount in the past, but it makes no difference that I noticed.

Cheers,

Scott.

[mitchelS]

Hi Scott,

thanks I really appreciate it.

The bioruptor seems to work really well. I sheared my DNA (5-10KB) on the medium setting at 10sec intervals for 10min. The resulting size ranged from 90-450bp with the main size being 190-300bp.

Seems good so far.

Cheers,

Mitch.