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new DNA protocol with really high titer lysates

| posted 17 Nov, 2015 02:10
We did the new DNA purification protocol (i.e. no PEG) with five students Thursday and results were kind of mixed, which by itself I can totally live with given that we normally got mixed results with the old prep anyway and mixed results that only take one day to get beats basically the same thing that takes two days plus requiring a gunky reagent! But here's my question:

At really high titers (ten to the tenth and above) do you all recommend that we really and truly dilute all the way down to say the middle of the ten to the ninth range?

The best result we got Thursday was from a young lady whose phage titered out in the e10 range: she was able to make about 15 micrograms

Then there were two in about the e11 range who made about 8 or 9 micrograms.

The two who were in about the e12 range made rather little: a total of maybe five or six micrograms, tops. Nobody had any notable quantity of DNA in their second or third tubes (all per nanodrop).

The e10 sample we used straight; e11 we diluted 1/1; e12 we diluted 1/5. I did this based on a piece of advice I got several years ago relative to the old protocol from Lu Barker, who advised against diluting anything beyond about 1/5.

Any advice?
| posted 17 Nov, 2015 18:44
I have never diluted any of my lysates prior to extracting DNA using the new protocol. In my experience, the higher the lysate, the higher the yield. If you are getting really low yields you can try adding two steps. First, you can add 15 uL of 0.5 molar EDTA to the sample following the 30 minute nuclease incubation. This will inactivate most of the nucleases. Then you can incubate the lysate at 65 degrees for 15 minutes to start degrading the capsid. Then you can proceed with the protocol and add the DNA clean up resin.

I hope this helps!
| posted 17 Nov, 2015 22:49
Marianne, the thing is that our yield problem is not across the board, and seems to be titer-related. I would think if it were a nuclease issue then the young lady with the e10 titer would be just as much affected as anybody, if not moreso. Plus I distinctly remember reading in Welkin's version of the protocol not to go above e11.
| posted 17 Nov, 2015 22:52
I'm also not using the qiagen nucleases, instead we're using 9 microliters of the nuclease mix from the old protocol. Not sure why I picked 9, but I figured if in the old protocol 2ml of resin could inactivate 40 lambda it could inactivate less than a fourth of that.
| posted 17 Nov, 2015 23:33
In my experience the low yields are not directly related to the nucleases, but to issues with the guanadinium to open the capsid. The heating really seems to combat this, but you have to inactivate the nucleases with the EDTA first. In addition, I think the advice to dilute high titers was specific for the phage precipitation protocol. We find that we can not get good yields with any titers less than 10^9.

I hope this helps!
| posted 17 Nov, 2015 23:55
I think I'm starting to see the light………thanks
| posted 22 Mar, 2019 18:36

This is my first time running SEA-PHAGES and we have some 10^11 and 10^12 titers. I saw this post but I wasn't sure if we needed to dilute them first because I remember at a meeting someone mentioning that it needed to be diluted if the titer was really high. I can't find it in the protocol now but I remember it being talked about a lot. Suggestions? Just try it and see what happens or should I try diluting it?

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