3 replies to this topic

[bitpas]

Hello all!

I have problems with a ligation reaction / transformation.
For the backbone Vector (13kb) i use 2 relativley close restriction sites:
etcNNNascITTAAsalINNNetc, sequential digest (asc first), the insert is 1.2kb.....      and the transformation just dont work!!!!


do you think the sites are too close? or other suggestions? (Ligase and competent cells are fine, vector:insert ratio from 1:1 to 1:5 all tried, Ligationreaction ON16°..)

Every help is desperatly welcome!

[lyrezxl]

Quote

do you think the sites are too close?

i don't think so. it always works well even the one just follow the other.

the backbone Vector (13kb), which is not like the small one , is difficult to estimate the DNA's amount accurately.  and that will affect the estimate of the V/I ratio. i think that's why it works not good.
is it a plant expression vector??
it always works like this.
you'd better just try it again and add more vectors, higher concentration of DNA, and the ratio should be 1:8 or more.

hope it helps.

lyrezxl

[tfitzwater]

Sal I is notorious for requiring a huge amount of overhanging sequence for efficient restriction activity.  Check the tables in the back of the New England Biolabs catalog.  People on the various biotech forums are constantly complaining of cloning difficulties when using Sal I.  In my hands, it is possible to use Sal I, but you have to pay attention to its quirks.  

Cut with Sal I first.  Note that supercoiled pBR322 or pUC require 10-fold more enzyme than linear lambda DNA.  (10 Units/ug of DNA)

When the Sal I cut is complete, perform a buffer exchange and add Asc I.  Asc I will cut with >90% efficiency without any overhanging sequence.  

The buffer requirements for these enzymes are quite different.  Asc I requires NEB Buffer 4.  Sal I requires more salt than Asc I.  Sal I will cut in Buffer 4 only when it is supplemented with NaCl to a final 100 mM concentration.  But then it is too salty for the Asc I.  So cut with Sal I, clean up the DNA and then cut with Asc I.

[Leyre]

Hi!
See what I said to the topic "ligation again" maybe I can help you.

Leyre