l******o
发帖数: 103 | 1 I have the following problem. I want to introduce a point mutation to my
plasmid. The plasmid is super big. So I first cut out a fragment(~4kb) with
XbaI and then ligate it to another vector. After introducing mutation with
mutagenesis kit, the sequencing result is as expected.
Next step is to put the XbaI-XbaI fragment containing the mutation back
into the original clone. After ligation and transformation with DH5alpha I
get a number of colonies (~20) but no colonies in the negative control. By
PCR with a internal primer& vector primer I did confirm the fragment is in
the plasmid. Meanwhile, the orientation can also be confirmed by this PCR.
So I midi-prep DNA and test the expression. Unfortunately, no expression at
all (positive control works well).
So I suspect some sort of genomic recombination took place in the Ecolis
since this plasmid is very big. Then the whole cDNA is not in frame. But I
never experienced this problem. If anyone of you experienced a similar
problem and has has any suggestions as to how to address this problem, your
help is greatly appreciated.
Will it reduce the recombination if I grow Ecoli in low temperature(~30
Celcius)?
Thank you!! |
o****u
发帖数: 214 | 2 这个挺常见的。特别是做了突变后，有可能导致质粒带毒。所以e.coli会启动某种
rescue，然后你的质粒就被搞的乱七八糟的了。
我没有什么好的方法解决。只有case by case的一点点试条件。祝好运。
with
at
【在 l******o 的大作中提到】
: I have the following problem. I want to introduce a point mutation to my
: plasmid. The plasmid is super big. So I first cut out a fragment(~4kb) with
: XbaI and then ligate it to another vector. After introducing mutation with
: mutagenesis kit, the sequencing result is as expected.
: Next step is to put the XbaI-XbaI fragment containing the mutation back
: into the original clone. After ligation and transformation with DH5alpha I
: get a number of colonies (~20) but no colonies in the negative control. By
: PCR with a internal primer& vector primer I did confirm the fragment is in
: the plasmid. Meanwhile, the orientation can also be confirmed by this PCR.
: So I midi-prep DNA and test the expression. Unfortunately, no expression at
|
h***e
发帖数: 146 | 3 先测个序吧
30度会减少重组
或者用其他的E。 coli Host |
n********k
发帖数: 2818 | 4 make sure it is what you said, if yes, may consider to change bacteria
strains, look for those which can overexpression long repeats DNA, or
inverted repeats, or for retro and lenti viral stuff...essentially those
lost RecABCD, not the only the RecA as the typical strains..if it is true,
this kind of cloning work is not trivial until you figure out right
materials...Or you could beat the nature by brutal force, I heard a guy
picked 20K clones to get one construct/plasmid done, that was before we know
the long repeats DNA cloning problems(bateria people always knew it much
earlier though)...I was completely dumbfounded! But it worked out for him
and beautifully...
with
at
【在 l******o 的大作中提到】
: I have the following problem. I want to introduce a point mutation to my
: plasmid. The plasmid is super big. So I first cut out a fragment(~4kb) with
: XbaI and then ligate it to another vector. After introducing mutation with
: mutagenesis kit, the sequencing result is as expected.
: Next step is to put the XbaI-XbaI fragment containing the mutation back
: into the original clone. After ligation and transformation with DH5alpha I
: get a number of colonies (~20) but no colonies in the negative control. By
: PCR with a internal primer& vector primer I did confirm the fragment is in
: the plasmid. Meanwhile, the orientation can also be confirmed by this PCR.
: So I midi-prep DNA and test the expression. Unfortunately, no expression at
|
r****r
发帖数: 36 | 5 1. use C41 E. coli, which is BL21 derivative. remember to use plasmid
purification kit that can eliminate ENDONUCLEASE. Due to its BL21 background
, the copy number of your plasmids would be kept relatively low and thus the
"toxicity" might pose less negative effects on right colonies. C41 is also
used to express "toxic" proteins very often.
2. use lower antibiotics in 2YT plates for C41.
3. incubate at room-temperature. Large and toxic plasmids always take 2 days
to form colonies. Colonies appeared overnight always contain wrong plasmids.
btw, did u check the integrity of your plasmid? |
n********k
发帖数: 2818 | 6 thank you!!
Question1: So large plasmid always behave this way or not? the biggest one I
worked was 20kb, it was ok with typical condition.
Question2: so cosmids or Yac/Bac don't follow the same way as they have
multiple rep origisns, right?
background
the
also
days
plasmids.
【在 r****r 的大作中提到】
: 1. use C41 E. coli, which is BL21 derivative. remember to use plasmid
: purification kit that can eliminate ENDONUCLEASE. Due to its BL21 background
: , the copy number of your plasmids would be kept relatively low and thus the
: "toxicity" might pose less negative effects on right colonies. C41 is also
: used to express "toxic" proteins very often.
: 2. use lower antibiotics in 2YT plates for C41.
: 3. incubate at room-temperature. Large and toxic plasmids always take 2 days
: to form colonies. Colonies appeared overnight always contain wrong plasmids.
: btw, did u check the integrity of your plasmid?
|
r****r
发帖数: 36 | 7 1. It depends on what genes were in your plasmids. for example, it is ok to
manipulate some DNA viruses with large genome using conventional cloning
method but when it comes to some RNA viruses, you can't even clone its
fragments more than 3K.
2. I have no experience on Cosmid/YAC/BAC.
I
【在 n********k 的大作中提到】
: thank you!!
: Question1: So large plasmid always behave this way or not? the biggest one I
: worked was 20kb, it was ok with typical condition.
: Question2: so cosmids or Yac/Bac don't follow the same way as they have
: multiple rep origisns, right?
:
: background
: the
: also
: days
|
e****s
发帖数: 1125 | 8 I would check plasmid and expression more carefully before draw a conclusion
of genomic recombination.
1. Have you check the whole 4kb cDNA after site-direct mutagenesis? It will
take you 5 sequencing to check the whole cDNA.
2. How do you check the expression? Have you check both supernatant and
total lysis? by western or just SDS-PAGE. The point is single mutant could
dramatically decrease the expression.
3. Purify plasmid from the expression E.coli, do cutting test and send for
sequencing.
with
at
【在 l******o 的大作中提到】
: I have the following problem. I want to introduce a point mutation to my
: plasmid. The plasmid is super big. So I first cut out a fragment(~4kb) with
: XbaI and then ligate it to another vector. After introducing mutation with
: mutagenesis kit, the sequencing result is as expected.
: Next step is to put the XbaI-XbaI fragment containing the mutation back
: into the original clone. After ligation and transformation with DH5alpha I
: get a number of colonies (~20) but no colonies in the negative control. By
: PCR with a internal primer& vector primer I did confirm the fragment is in
: the plasmid. Meanwhile, the orientation can also be confirmed by this PCR.
: So I midi-prep DNA and test the expression. Unfortunately, no expression at
|
l******o
发帖数: 103 | 9 Thank you all for the kind reply.
1. I did not check the whole 4kb cDNA after site-direct mutagenesis. I sent
it for sequencing yesterday.
2. After screening positive colonies by PCR, I midi-prep DNA and transfect
into mammalian cells. After 24h, cells were harvested and directly boiled
with sample buffer. I run SDS-PAGE and probed the membrane with Ab against
the epitope. The original clone got good expression.
3. I cut the original clone with XbaI (see the following agarose gel photo).
The 4kb DNA can be released successfully. But it is very weird with the new
construct.
conclusion
will
【在 e****s 的大作中提到】
: I would check plasmid and expression more carefully before draw a conclusion
: of genomic recombination.
: 1. Have you check the whole 4kb cDNA after site-direct mutagenesis? It will
: take you 5 sequencing to check the whole cDNA.
: 2. How do you check the expression? Have you check both supernatant and
: total lysis? by western or just SDS-PAGE. The point is single mutant could
: dramatically decrease the expression.
: 3. Purify plasmid from the expression E.coli, do cutting test and send for
: sequencing.
:
|
l******o
发帖数: 103 | 10 我想问的是，如果要减少重组，降低温度。涂板后就要放在低温（～30）incubate？还
是从liquid culture那步才需要30度？
background
the
also
days
plasmids.
【在 r****r 的大作中提到】
: 1. use C41 E. coli, which is BL21 derivative. remember to use plasmid
: purification kit that can eliminate ENDONUCLEASE. Due to its BL21 background
: , the copy number of your plasmids would be kept relatively low and thus the
: "toxicity" might pose less negative effects on right colonies. C41 is also
: used to express "toxic" proteins very often.
: 2. use lower antibiotics in 2YT plates for C41.
: 3. incubate at room-temperature. Large and toxic plasmids always take 2 days
: to form colonies. Colonies appeared overnight always contain wrong plasmids.
: btw, did u check the integrity of your plasmid?
|
r****r
发帖数: 36 | 11 Keep at RT in all steps.
BTW, from your gel image, it seems like your XbaI sites were gone in new
construct.
【在 l******o 的大作中提到】
: 我想问的是，如果要减少重组，降低温度。涂板后就要放在低温（～30）incubate？还
: 是从liquid culture那步才需要30度？
:
: background
: the
: also
: days
: plasmids.
|
e****s
发帖数: 1125 | 12 I see.
Apparently, your new constructs are just not right based on your digestion
tests, which explains the expression problem.
Digestion tests is always better than PCR to confirm the subcloning
constructs.
I only sequence samples after digestion tests.
Sometimes I struggle with the ligation of big constructs as well.
Good luck!
sent
).
new
【在 l******o 的大作中提到】
: Thank you all for the kind reply.
: 1. I did not check the whole 4kb cDNA after site-direct mutagenesis. I sent
: it for sequencing yesterday.
: 2. After screening positive colonies by PCR, I midi-prep DNA and transfect
: into mammalian cells. After 24h, cells were harvested and directly boiled
: with sample buffer. I run SDS-PAGE and probed the membrane with Ab against
: the epitope. The original clone got good expression.
: 3. I cut the original clone with XbaI (see the following agarose gel photo).
: The 4kb DNA can be released successfully. But it is very weird with the new
: construct.
|
r****r
发帖数: 36 | 13 Indeed, it's more difficult to manipulate big constructs than small
constructs with ligation method. So sometimes I used yeast recombination to
handle big constructs. The drawback of using yeast is the high mutation rate
. I always have to use several independent clones to confirm my observation
was really due to my mutations instead of random mutations caused by yeast.
【在 e****s 的大作中提到】
: I see.
: Apparently, your new constructs are just not right based on your digestion
: tests, which explains the expression problem.
: Digestion tests is always better than PCR to confirm the subcloning
: constructs.
: I only sequence samples after digestion tests.
: Sometimes I struggle with the ligation of big constructs as well.
: Good luck!
:
: sent
|
l******o
发帖数: 103 | 14 problem solved!!!! I got the right construct!!!!
Every steps were performed in 30 degree except heat shock (42 degree).
Thank you all!!!! |
