Gibson assembly - 未名空间MITBBS历史存档

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Gibson assembly

Gibson assembly# Biology - 生物学

b*t2015-08-07 07:08

1 楼

H*N2015-08-07 07:08

2 楼

Can anybody share you experience with Gibson assembly? Planning to do it,
but would like to hear your tips and potential pitfalls with the technique.
Thanks in advance.

a*n2015-08-07 07:08

3 楼

I just followed the product instruction and succeeded. I used mixture from
NEB.

【在 H****N 的大作中提到】

: Can anybody share you experience with Gibson assembly? Planning to do it,
: but would like to hear your tips and potential pitfalls with the technique.
: Thanks in advance.

w*e2015-08-07 07:08

4 楼

me 2

【在 a*****n 的大作中提到】

: I just followed the product instruction and succeeded. I used mixture from
: NEB.

H*N2015-08-07 07:08

5 楼

Thanks to wne and ankyin. How large was your final product? How many
fragments did you use?

a*n2015-08-07 07:08

6 楼

I just cloned a 1.5kb cdna with a tag into a regular vector.

【在 H****N 的大作中提到】

: Thanks to wne and ankyin. How large was your final product? How many
: fragments did you use?

M*g2015-08-07 07:08

7 楼

Just use NEB gibson assembly kit or their newer version NEBuilder hifi DNA
assembly kit. you can clone up to 6 fragments. Make sure use their
competent cells, since the assembly rxn many times is not compatible with
competent cells from other companies such as invitrogen or homemade cells.
you can use NEBuilder software to design your primers: http://nebuilder.neb.com/.
I have assembled 4 fragments and plasmids are normally 7-8 kb. if you have
questions, just call NEB tech support, and they are helpful. that's what i
normally did.
hope this helps.

w*e2015-08-07 07:08

8 楼

I cloned 3 fragments together (each is 2k) and it works.

【在 H****N 的大作中提到】

: Thanks to wne and ankyin. How large was your final product? How many
: fragments did you use?