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】
: 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】
: 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】
: 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】
: 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】
: 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】
: 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】
: 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】
: 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】
: 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 | |
|
