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Low Miniprep Yield

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#1 TVo



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Posted 01 August 2011 - 11:03 PM

Hey board,

I've recently been trying to miniprep pEntry vectors using home-made P1, P2, N3 buffers, filter/binding plates, etc. etc. (for the purpose of Gateway cloning). Previously, I've been able obtain quite a decent plasmid yield (~100 ng/uL). But it's been 7 months since my last miniprep spree of these vectors and, suddenly it seems, my yield has been very low (15-50 ng/uL). All of my buffers were freshly made except for P1 which was made ~7 months ago and did not have RNase A added until very recently (and stored at 4C). I tried to vary my DH5 alpha inoculation times between 16 and 28 hours with very little change in yield (previously when the yields were good, I inoculated anywhere from 18 to 20 hours). I always inoculate my bacteria in SOC media (should be richer in nutrients than LB) with kanamycin antibiotics.

Can it be possible that my P1 solution without RNase A and stored at 4C for 7 months got bad and somehow ruined the minipreps?

*Also, I do not reuse filter/binding plates or buffers. The buffers were made according to Qiagen's recipe or somewhere else I found. And I don't think it's due to variation in my pipetting...since I automate my minipreps. I just script something and a robot does it for me. =)

Edited by TVo, 01 August 2011 - 11:30 PM.

#2 bob1


    Thelymitra pulchella

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Posted 02 August 2011 - 05:33 PM

It is possible that your P1 is off, though this is usually just salt, a buffer and some glucose. It may be that your NaOH has precipitated, if you have a stock solution, try preparing fresh and see how it goes.

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