Posted 05 May 2004 - 11:57 PM
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!
Posted 06 May 2004 - 05:41 PM
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.
Posted 12 May 2004 - 03:35 PM
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.
Posted 17 May 2004 - 12:05 AM
See what I said to the topic "ligation again" maybe I can help you.