f****h 发帖数: 41 | 1 I have a question to ask people who know molecular biology. I have a plasmid
in DH5a cells and I can get 10ug DNA (200ng/uL, 50uL) from 1.5mL miniprep
culture. However, when I cultured 250mL for maxiprep, I only got 130ug DNA,
which is more than 10 times less than what I expected. The procedure of
extracting DNA should be fine because I got 900ug DNA from another plasmid
in parallel.
Can anyone give me some comments/suggestions? Thank you so much! | f***4 发帖数: 886 | 2 这次细菌养的有些老了?
plasmid
,
【在 f****h 的大作中提到】![](/moin_static193/solenoid/img/up.png) : I have a question to ask people who know molecular biology. I have a plasmid : in DH5a cells and I can get 10ug DNA (200ng/uL, 50uL) from 1.5mL miniprep : culture. However, when I cultured 250mL for maxiprep, I only got 130ug DNA, : which is more than 10 times less than what I expected. The procedure of : extracting DNA should be fine because I got 900ug DNA from another plasmid : in parallel. : Can anyone give me some comments/suggestions? Thank you so much!
| f****h 发帖数: 41 | 3 I tried three times, 16hrs, 15hrs, 14hrs, the results are between 90-150ug.
【在 f***4 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 这次细菌养的有些老了? : : plasmid : ,
| f***4 发帖数: 886 | 4 我觉得还是细菌的问题。是从单菌落/过夜菌/大瓶 来的吗?
【在 f****h 的大作中提到】![](/moin_static193/solenoid/img/up.png) : I tried three times, 16hrs, 15hrs, 14hrs, the results are between 90-150ug.
| f****h 发帖数: 41 | 5 I picked up a single colony in the morning and grew it in 1.5mL culture from
9AM to 6PM, then from this miniculture, I took 250uL into 250mL, grew
overnight.
I thought about the bacteria, should I change the DH5a to a different one?
【在 f***4 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 我觉得还是细菌的问题。是从单菌落/过夜菌/大瓶 来的吗?
| f***4 发帖数: 886 | 6 这个不太懂了,试试吧
from
【在 f****h 的大作中提到】![](/moin_static193/solenoid/img/up.png) : I picked up a single colony in the morning and grew it in 1.5mL culture from : 9AM to 6PM, then from this miniculture, I took 250uL into 250mL, grew : overnight. : I thought about the bacteria, should I change the DH5a to a different one?
| j****x 发帖数: 1704 | 7 这个问题在本版应该就讨论过不下三遍了,好消息是你绝不是第一个遇到类似问题的人
,坏消息是可能没有特别的解决办法。
我记得之前有一例是,换过菌株,换过培养条件(培养基温度时间什么的),换过抽提
方法,但是maxi就是不正常,最后只好大量小提然后再合并。
你可以试着换一换各种条件,或者试试看midi,也许有用也许没用,试过才知道。好运!
plasmid
,
【在 f****h 的大作中提到】![](/moin_static193/solenoid/img/up.png) : I have a question to ask people who know molecular biology. I have a plasmid : in DH5a cells and I can get 10ug DNA (200ng/uL, 50uL) from 1.5mL miniprep : culture. However, when I cultured 250mL for maxiprep, I only got 130ug DNA, : which is more than 10 times less than what I expected. The procedure of : extracting DNA should be fine because I got 900ug DNA from another plasmid : in parallel. : Can anyone give me some comments/suggestions? Thank you so much!
| s********s 发帖数: 67 | 8 我也遇到过这样的问题,后来发现是摇的时间太短了。后来摇24小时或者更长反而好些
。现在一般先取一点小提看一下,算一下量,然后再大提。
plasmid
,
【在 f****h 的大作中提到】![](/moin_static193/solenoid/img/up.png) : I have a question to ask people who know molecular biology. I have a plasmid : in DH5a cells and I can get 10ug DNA (200ng/uL, 50uL) from 1.5mL miniprep : culture. However, when I cultured 250mL for maxiprep, I only got 130ug DNA, : which is more than 10 times less than what I expected. The procedure of : extracting DNA should be fine because I got 900ug DNA from another plasmid : in parallel. : Can anyone give me some comments/suggestions? Thank you so much!
| f****h 发帖数: 41 | 9 Ok, I will change the bacteria strain and tell you guys what happen. Thanks!
运!
【在 j****x 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 这个问题在本版应该就讨论过不下三遍了,好消息是你绝不是第一个遇到类似问题的人 : ,坏消息是可能没有特别的解决办法。 : 我记得之前有一例是,换过菌株,换过培养条件(培养基温度时间什么的),换过抽提 : 方法,但是maxi就是不正常,最后只好大量小提然后再合并。 : 你可以试着换一换各种条件,或者试试看midi,也许有用也许没用,试过才知道。好运! : : plasmid : ,
| j****x 发帖数: 1704 | 10 如果要摇超过16个小时,有必要加氯霉素,否则即使能提出质粒来质量也不会好的
【在 s********s 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 我也遇到过这样的问题,后来发现是摇的时间太短了。后来摇24小时或者更长反而好些 : 。现在一般先取一点小提看一下,算一下量,然后再大提。 : : plasmid : ,
| T******y 发帖数: 14506 | |
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