j*****q
发帖数: 82 | 1 大大们进来看看
两条homo arm一定要一模一样的序列吗？
如果其中一条（长的那条，大概6kbp）中间有大概100bp不到的不同序列，会影响
recombination的efficiency吗？
多谢 |
n********k
发帖数: 2818 | 2 it depends on what you are doing, and how big the risk u like to take?
Nobody really knows in practice(I
asssume u are doing mice knockout?......for easy targeting locus, it is
likely fine but if you are unlucky, u
might get nothing...if it is easy to correct, I would just remake it,
otherwise go ahead with your targeting and
see what happens...If it were me, I'll definitely remake the construct, it
takes nothing to remake one:))
compare to the targeting,. one step is about a week work, isn't it
【在 j*****q 的大作中提到】
: 大大们进来看看
: 两条homo arm一定要一模一样的序列吗？
: 如果其中一条（长的那条，大概6kbp）中间有大概100bp不到的不同序列，会影响
: recombination的efficiency吗？
: 多谢
|
n********k
发帖数: 2818 | 3 BTw, there are several experts on this board on the topic, maybe they will
have different opinion....
【在 j*****q 的大作中提到】
: 大大们进来看看
: 两条homo arm一定要一模一样的序列吗？
: 如果其中一条（长的那条，大概6kbp）中间有大概100bp不到的不同序列，会影响
: recombination的efficiency吗？
: 多谢
|
j*****q
发帖数: 82 | 4 多谢ls的。
这100bp不到的序列是一定要加的，类似一个mutation。HR的目的是knock in。因为就
100bp不到的sequences，我就直接做在一个homo arm里了。不知道可不可以。
继续等大大 |
j*****q
发帖数: 82 | |
n********k
发帖数: 2818 | 6 it is a totally different question now...u are talking about two
recombination events or hope to have one non-
perfect recombination event so that u can get in both ur knockin arm and
another 100bp with ur long arm...I
don't know, it could be tricky..I have a friend who did similar things but
used double selections... don't really
have experience with this, so maybe someone else could help...BTW, u could
have asked question more
clearly...no biggie
【在 j*****q 的大作中提到】
: 多谢ls的。
: 这100bp不到的序列是一定要加的，类似一个mutation。HR的目的是knock in。因为就
: 100bp不到的sequences，我就直接做在一个homo arm里了。不知道可不可以。
: 继续等大大
|
H****N
发帖数: 997 | 7 It should work. Just be aware that you will have two possible recombination
events: in one event, the middle fragment is used and therefore you
insertion will not be introduced; in another, the fragment on the side is
used and you will have the desired allele with you insertion. You have
to have a genotyping strategy that can distinguish these two events. You may
also increase the length of the homologous fragment on the side to maximize
your chance of success. This is actually very similary to |
n********k
发帖数: 2818 | 8 Finally u came:)))...I also have a question in this case, so will the
efficiency of desired targeting much lower, or
not really with your experience... Another question is what if the middle
fragment is like 10kb or even more....I
was thinking it would take two recombination events to get them in...then
the efficiency would be really low...
recombination
may
maximize
floxed
【在 H****N 的大作中提到】
: It should work. Just be aware that you will have two possible recombination
: events: in one event, the middle fragment is used and therefore you
: insertion will not be introduced; in another, the fragment on the side is
: used and you will have the desired allele with you insertion. You have
: to have a genotyping strategy that can distinguish these two events. You may
: also increase the length of the homologous fragment on the side to maximize
: your chance of success. This is actually very similary to
|
H****N
发帖数: 997 | 9 Hey how is it going? To your first question, the efficiency will be lower,
but not that much lower. Say if you have 3 kb on the side and 2kb in the
middle, you may have half the efficiency of what you would without the
insertion. As to the second question, if you have a 10kb fragment in the
middle, the chance of getting the desired recombination may be very low. It'
s just my gut feeling and I don't have any actual experience with a long
middle arm. If I were you, I would give it a try, if the
【在 n********k 的大作中提到】
: Finally u came:)))...I also have a question in this case, so will the
: efficiency of desired targeting much lower, or
: not really with your experience... Another question is what if the middle
: fragment is like 10kb or even more....I
: was thinking it would take two recombination events to get them in...then
: the efficiency would be really low...
:
: recombination
: may
: maximize
|
n********k
发帖数: 2818 | 10 thanks a lot, don't have anything like that in planning , just something I
thought i shall ask your opinion about
it in case I need to do in the future...so far everything is on track,
finished one study last year and trying to
wrap another one this year and then hope to have more freedom to think about
moving on...
,
It'
【在 H****N 的大作中提到】
: Hey how is it going? To your first question, the efficiency will be lower,
: but not that much lower. Say if you have 3 kb on the side and 2kb in the
: middle, you may have half the efficiency of what you would without the
: insertion. As to the second question, if you have a 10kb fragment in the
: middle, the chance of getting the desired recombination may be very low. It'
: s just my gut feeling and I don't have any actual experience with a long
: middle arm. If I were you, I would give it a try, if the
|
|
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j*****q
发帖数: 82 | 11 hi, henben,
yes, the less than 100bp contains a loxp site. acutally i am also aware of
two HR event in it. but i dnt have any marker to select the 100bp events.
but it is quite easy to distinguish the insertion.
does it mean the second HR to integrate the 100bp will be very low
efficiency?
does it mean the introduction of 100bp will be just by chance? |
j*****q
发帖数: 82 | 12 and Happy new year all guys. |
F******p
发帖数: 2099 | 13 中间什么位置？
【在 j*****q 的大作中提到】
: 大大们进来看看
: 两条homo arm一定要一模一样的序列吗？
: 如果其中一条（长的那条，大概6kbp）中间有大概100bp不到的不同序列，会影响
: recombination的efficiency吗？
: 多谢
|
j*****q
发帖数: 82 | 14 3k+5k的位置。
does it matter？ |
H****N
发帖数: 997 | 15 Sorry I did not see this earlier. You don't need a selection marker for the
insertion, but you need a genotyping strategy to distinguish the two HR
events.
【在 j*****q 的大作中提到】
: hi, henben,
: yes, the less than 100bp contains a loxp site. acutally i am also aware of
: two HR event in it. but i dnt have any marker to select the 100bp events.
: but it is quite easy to distinguish the insertion.
: does it mean the second HR to integrate the 100bp will be very low
: efficiency?
: does it mean the introduction of 100bp will be just by chance?
|
s******y
发帖数: 28562 | 16 For yeast, it will be alright.
For mammalian system...I don't know
【在 j*****q 的大作中提到】
: 多谢ls的。
: 这100bp不到的序列是一定要加的，类似一个mutation。HR的目的是knock in。因为就
: 100bp不到的sequences，我就直接做在一个homo arm里了。不知道可不可以。
: 继续等大大
|
