[PStepper]

Hi everyone,

this is the main discussion space for BOMB protocol #7.1 gDNA extraction.
Here you can post all your questions and feedback!

[PStepper]

reserved space

[AKSchilling]

Hi,

thanks for your helpful protocols. Do you have or working at a protocol for gDNA extraction for insects?

[Tomek]

Dear AKSchiling,

We never tried extracting NA from insects. I suppose this should not be too difficult to optimize. Probably you could follow the “Tissue extraction” protocols, where you would release the NA with ProtK treatment and then GITC buffer for cell lysis and NA capture.

Cheers,

Tomek

[TimH]

Hi AK Schilling – sorry for the slow reply – in case you are still wondering whether to give it a go: yes, people in my Department have tried BOMB on insects, following the Tissue extraction protocol as suggested by Tomek. I don’t think the chitin exoskeleton was broken down, but DNA was extracted all the same.

Cheers,

Tim

[Audrey McDonald]

Hi, thanks so much for all the information and protocols!

Do you have any recommendations for modifying the gDNA extraction protocol for plant tissue extractions?

[Phil Oberacker]

Hey Audrey,

there is a protocol for plant tissue TNA extraction available (#6.4). The protocol also contains a gDNA version, as you simply do an RNase digest after lysis.

Cheers
Phil

[Jon Sanders]

In your paper, you mention the possibility of substituting in commercially prepared magnetic particles (such as these). We would like to try these out. How exactly are you preparing them? Presumably they are diluted somehow?

[TimH]

Hi Jon – to prepare those beads for gDNA, RNA and TNA extraction take 1 mL of thoroughly resuspended bead mixture and place on a magnetic. Once beads have settled, remove the storage buffer (it contains sodium azide) and resuspend in 1 mL of TE buffer off the magnetic. Repeat process at least twice more and then resuspend in TE. We do this into 50 mL of TE – seems to be a good concentration of beads, however this could likely be altered without making much difference.

Cheers, Tim

[Nuria]

Hi,

Thank you for sharing these efficient protocols. I am doing gDNA extraction from gonads (ovaries and testis) with your protocol (6.3). However, I am getting low 260/230 ratios. What would you recommend?

Cheers,

[admin]

Ahoy,

As the low 260/230 might be the result of GITC being carried over into to the final elution the first thing you should try is performing one or two additional 80% EtOH wash steps.

Cheers

Tim M

• This reply was modified 4 years, 1 month ago by admin.

[Jennifer Kuehl]

Hello

I am also getting very low 260/230 ratios.  I tried up to eight 80% ethanol washes, the ratios  improve with more washing but  they are still low ~.6 for gram positive bacteria and 1.7 for gram negative.  I am considering  a larger volumes of 70% ethanol (600ul) for the wash steps or perhaps a secondary bead clean-up.  Any recommendations?

[admin]

Not really, at least nothing you haven’t suggested yourself. I frequently use 70% EtOH for my washes so that should work but if it will solve you issue I cannot tell.  Are you making sure there is no liquid transferred from wash to wash inside the tube lid? Cheers

[Emilly]

Hello!

I was reading your protocol of gDNA extraction with beads and a got confused with ratio mentioned 2:3:4. How is it used with blood sample and without TE buffer at beggining? It’s 140ul of blood and 20ul magnetic bead?

Best regards.

[TimH]

Hi Emily,

Blood does not really work that well with the 2:3:4 ratio, as there is a lot of liquid in there and (for humans/mammals) not many nucleated cells. You are probably better off going with a 2-step ’tissue’ protocol (#6.3), where you first protK treat in TNES, and then add in 1.5X GITC. From this point on it is basically 2:3:4.

Best wishes, Tim H

 

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