SEA-PHAGES | DNA Isolation from M. foliorum

Link to this post \| posted 13 Oct, 2018 02:47
viknesh	c.sunnen Sorry to keep bugging, but I'm revising our protocol: what's the concentration of Proteinase K? The instructor guide protocol calls for 5uL per 1mL sample, but it doesn't have a concentration. Also, you recommended above 0.5M EDTA, but the protocol says 0.1M. Is one concentration preferred. Thank-you again for all of your input, and thanks Maria for your insight! If we have trouble, we'll probably eliminate the nuclease step, too! Nikki Thanks for pointing that out. The concentrations were only included in the Discovery Guide, and not in the Instructors Guide. It's now been updated.

Link to this post | posted 13 Oct, 2018 02:47

c.sunnen
Sorry to keep bugging, but I'm revising our protocol: what's the concentration of Proteinase K? The instructor guide protocol calls for 5uL per 1mL sample, but it doesn't have a concentration. Also, you recommended above 0.5M EDTA, but the protocol says 0.1M. Is one concentration preferred.

Thank-you again for all of your input, and thanks Maria for your insight! If we have trouble, we'll probably eliminate the nuclease step, too!

Nikki

Thanks for pointing that out. The concentrations were only included in the Discovery Guide, and not in the Instructors Guide. It's now been updated.

Link to this post \| posted 14 Oct, 2018 16:34
viknesh	c.sunnen Sorry to keep bugging, but I'm revising our protocol: what's the concentration of Proteinase K? The instructor guide protocol calls for 5uL per 1mL sample, but it doesn't have a concentration. Also, you recommended above 0.5M EDTA, but the protocol says 0.1M. Is one concentration preferred. Thank-you again for all of your input, and thanks Maria for your insight! If we have trouble, we'll probably eliminate the nuclease step, too! Nikki I edited my post above to reflect the correct concentration (should someone else refer to it).

Link to this post | posted 14 Oct, 2018 16:34

viknesh

c.sunnen
Sorry to keep bugging, but I'm revising our protocol: what's the concentration of Proteinase K? The instructor guide protocol calls for 5uL per 1mL sample, but it doesn't have a concentration. Also, you recommended above 0.5M EDTA, but the protocol says 0.1M. Is one concentration preferred.

Thank-you again for all of your input, and thanks Maria for your insight! If we have trouble, we'll probably eliminate the nuclease step, too!

Nikki

I edited my post above to reflect the correct concentration (should someone else refer to it).

Link to this post \| posted 23 Oct, 2018 21:03
c.sunnen	Just thought I'd share that we got great DNA concentrations and quality today with our first attempt with this protocol. We only used 2mL (not the 5 our protocol says), and got concentrations around 250ng/ul, with A260/280 of 2-ish. Very pleased! Thanks for all your help! Now to run it on a gel with/without RE buffers… Nikki (I've attached our protocol here, in case anyone else wants to try it). 16Kb

Link to this post | posted 23 Oct, 2018 21:03

c.sunnen

Just thought I'd share that we got great DNA concentrations and quality today with our first attempt with this protocol. We only used 2mL (not the 5 our protocol says), and got concentrations around 250ng/ul, with A260/280 of 2-ish. Very pleased! Thanks for all your help! Now to run it on a gel with/without RE buffers…

Nikki
(I've attached our protocol here, in case anyone else wants to try it).

Link to this post \| posted 13 Nov, 2019 22:10
DrCatalase	Help, We got decent yields of DNA that and we tried to digest with NspI. the lanes are from two different phage lysates. Lanes: 1. DNA1 water 2. DNA1 water Buffer NspI (NEB 37 degrees 15 minutes) 3. DNA1 water Buffer 4. Ladder 5. DNA2 water 6. DNA2 water Buffer NspI (NEB 37 degrees 15 minutes) 7. DNA2 water Buffer We got no digestion except smearing in lane 6… we want to try again anyone have any suggestions? Thanks, Sean 90Kb

Link to this post | posted 13 Nov, 2019 22:10

DrCatalase

Help,
We got decent yields of DNA that and we tried to digest with NspI. the lanes are from two different phage lysates.
Lanes:
1. DNA1 water
2. DNA1 water Buffer NspI (NEB 37 degrees 15 minutes)
3. DNA1 water Buffer
4. Ladder
5. DNA2 water
6. DNA2 water Buffer NspI (NEB 37 degrees 15 minutes)
7. DNA2 water Buffer

We got no digestion except smearing in lane 6… we want to try again anyone have any suggestions?
Thanks,
Sean

Link to this post \| posted 14 Nov, 2019 03:22
c.sunnen	Hi! My first suggestion is to digest longer; we typically do ours for at least an hour at 37deg or overnight at room temp. Good news is your DNA looks nice! Some smearing, but mostly intact. If you're uncertain if your enzyme is any good, you can try digesting some lambda DNA (provided you have some on hand). I hope this is helpful! Good luck! Nikki P.S. If you're using the protocol I've posted above, there have been a few edits, though it should still work in its current form.

Link to this post | posted 14 Nov, 2019 03:22

c.sunnen

Hi!

My first suggestion is to digest longer; we typically do ours for at least an hour at 37deg or overnight at room temp. Good news is your DNA looks nice! Some smearing, but mostly intact. If you're uncertain if your enzyme is any good, you can try digesting some lambda DNA (provided you have some on hand).

I hope this is helpful! Good luck!
Nikki

P.S. If you're using the protocol I've posted above, there have been a few edits, though it should still work in its current form.

Link to this post \| posted 14 Nov, 2019 03:39
MSMC	DrCatalase Help, We got decent yields of DNA that and we tried to digest with NspI. the lanes are from two different phage lysates. Lanes: 1. DNA1 water 2. DNA1 water Buffer NspI (NEB 37 degrees 15 minutes) 3. DNA1 water Buffer 4. Ladder 5. DNA2 water 6. DNA2 water Buffer NspI (NEB 37 degrees 15 minutes) 7. DNA2 water Buffer We got no digestion except smearing in lane 6… we want to try again anyone have any suggestions? Thanks, Sean We've run into this issue before, it seems to be due to the conditions of the DNA sample after extraction - when we re-extracted using Phenol/Chloroform, we got digestion. It might be that you have either high concentration of salts or EDTA in the sample.

Link to this post | posted 14 Nov, 2019 03:39

MSMC

DrCatalase
Help,
We got decent yields of DNA that and we tried to digest with NspI. the lanes are from two different phage lysates.
Lanes:
1. DNA1 water
2. DNA1 water Buffer NspI (NEB 37 degrees 15 minutes)
3. DNA1 water Buffer
4. Ladder
5. DNA2 water
6. DNA2 water Buffer NspI (NEB 37 degrees 15 minutes)
7. DNA2 water Buffer

We got no digestion except smearing in lane 6… we want to try again anyone have any suggestions?
Thanks,
Sean

We've run into this issue before, it seems to be due to the conditions of the DNA sample after extraction - when we re-extracted using Phenol/Chloroform, we got digestion. It might be that you have either high concentration of salts or EDTA in the sample.

Link to this post \| posted 14 Nov, 2019 03:40
MSMC	Also, make sure you check with more than one enzyme. It's not uncommon for some enzymes to show few bands from digestion.

Link to this post \| posted 14 Nov, 2019 15:17
DrCatalase	Thank you all. I will try cleaning the DNA and digesting for longer. BTW I did the PEG protocol 9.2a from the instructor's guide. Edited 14 Nov, 2019 15:27

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DNA Isolation from M. foliorum - any updates?