s**x
发帖数: 138 | 1 i did ligation and transformation. many colonies were seen on dish. then i
picked up colonies to grow. nothing grows. i tried 3 times and the last time
i used freshly prepared LB medium. any1 teaches me what is going on? what i
should do next?? thx |
s**x
发帖数: 138 | 2 i am sure i used the right antibiotics |
f********n
发帖数: 6465 | 3 something wrong with PCR? |
s**x
发帖数: 138 | 4 but there are colonies on the plate with the same antibiotics. |
m**z
发帖数: 787 | 5 negative control for your plate? simply plate some cell to see whether they
can grow as well. this may rule out the possiblity of your LB plate being
bad...
time
i
【在 s**x 的大作中提到】
: i did ligation and transformation. many colonies were seen on dish. then i
: picked up colonies to grow. nothing grows. i tried 3 times and the last time
: i used freshly prepared LB medium. any1 teaches me what is going on? what i
: should do next?? thx
|
n***w
发帖数: 2405 | 6 you mean nothing grew... you mean after your miniprep, the product is empty?
or you mean the medium after couples of hours or overnight is still clear..
..? |
G***y
发帖数: 1082 | 7 2nd this. First check to make sure your LB plate is still good.The selection
strength might decrease if the plates were made a while ago.
they
【在 m**z 的大作中提到】
: negative control for your plate? simply plate some cell to see whether they
: can grow as well. this may rule out the possiblity of your LB plate being
: bad...
:
: time
: i
|
d****u
发帖数: 1553 | 8 你这个问题很好解决，假设你的实验每一步都是正确的。首先，你做连接的时候，一定
有一个载体的自连对照（只有vector，没有insert），说明这不是你的载体自连。其次
你做转化的时候首先确认抗性没有问题，同时做以下几个转化：
1，载体自连对照 （应该在盘上长非常非常少）
2，insert only （应该一个都不长）
3, 你的连接产物 （如果你的实验没问题，应该长很多）
4，找一个你以前用过的没问题的相同抗性的质粒 （你可以通过计算想长多少长多少）
5，找一个你以前用过的没问题的相同抗性的质粒 （你再怎么计算也一个都不该长）
然后你就知道问题出在哪里了。 |
h*********s
发帖数: 34 | 9 It occured to me that sometime transformed bacteria grows poorly in LB broth
, but grows well in LB plate. I figured out that it was because of the
protein expressed. The solution is to shake longer or try a midprep so that
you can have enough plasmid. |
T**********t
发帖数: 1604 | 10 How did you choose your colony to pick?
How much volume of LB did you inoculate with a single colony and what type
and concentration of antibiotics are you using?
How long did you grow your LB culture? What temperature and speed?
Some general thoughts without any details regarding the above questions.
1）pour fresh LB plates, make fresh LB media
2) repeat ligation and transformation, make sure to include positive and
negative control
3) check plates to make sure you have healthy colonies and your control
plates are normal. Pick single colonies of medium size. Don't pick tiny
colonies because they might be satellite colonies. Don't pick large colonies
either because they may be overgrown. Actually do not let your plate
overgrow, if your colonies grow fast, try lower the incubation temperature
by 3-5 degrees to slow them down.
4) make sure you use exact the same antibiotic and same concentration on the
LB plates and in the liquid LB media.
5) check the temperature setting of your incubator to see if it's accurate.
make sure your culture is agitated sufficiently during incubation. |
|
|
s******s
发帖数: 13035 | 11 有时候可能是很简单的原因，比如你接种针烧的太烫了。
time
i
【在 s**x 的大作中提到】
: i did ligation and transformation. many colonies were seen on dish. then i
: picked up colonies to grow. nothing grows. i tried 3 times and the last time
: i used freshly prepared LB medium. any1 teaches me what is going on? what i
: should do next?? thx
|
T**********t
发帖数: 1604 | 12 You can use sterile pipette tips.
【在 s******s 的大作中提到】
: 有时候可能是很简单的原因，比如你接种针烧的太烫了。
:
: time
: i
|
n***w
发帖数: 2405 | 13 VWR cell spreader 我刚买。。。呵呵。。。
【在 T**********t 的大作中提到】
: You can use sterile pipette tips.
|
d****u
发帖数: 1553 | |
s**x
发帖数: 138 | 15 the plates i used were made about 4 months ago. i think this is the reason.
i picked up 2 colonies and grew in lb broth without antibiotics. bacteria
grew. now i believe that the plates were not good. yesterday i repeated with
recently-made plates and there are colonies. i will grow bacteria tonight
to see what happens. thx for all suggestions. |
