Vector insertion for beginners - (Nov/11/2005 )
In my continuous process of learning, I have two new questions resquesting your expertise and experience.
I'm trying yo insert a 800bp sequence into pTarget Mammalian Vector.
Which is the DNA quantity (ng) I should start with? And the insert:vector ratio can vary from 3:1 to 1:3? Should I start with 3:1? I suppose if I play with this parameters I can improve my efficiency but if I get a good colony with the right construct then I can use plasmid preparations to get more amount and I can forget this insertion process, am I right?
Second question: I've been told that in order to save time and money, I should do sequencing of my insert after screening them with specifics primers, so I can be almost certain that the sequence will be good. Is this right?
Thank you very much for your help.
Always learning and improving.
The amount you start with is only important for the concentration of the ligation reaction, this should be about 20ng/ul
I think the range for insert to vector is 1:8 to 8:1
yes once you have a good colony you can stop with the ligation...
I agree, it saves money to check the insert yourself, screening by sequencing would be ridiculously expensive...
Thank you for your helpful response.
I have one last question. If you don't sequence your insert, how do you know that a mutation has been introduced which could lead to a protein with different properties.
First you can isolate DNA from some colonies 7- 20 (the number depends if you have high background or not). Do mini and digest them with the enzymes you used for cloning, then you know which clones contain the insert.
If you have high background (vector itself gives many colonies) then is better to do a colony PCR, to also check which colonies have the insert.
After this you can choose 3 or 4 of the clones you know they have the insert and sequence them.
I don't know how big is the insert but for small inserts, using a proof-reading enzyme, the sequence must be good. But I always sequence my mini's.
I think I mislead you, I meant that you should confirm your vector has the right insert some other way before you pay to have it sequenced -- you do still need to sequence the clone..