ligation/transformation issue - (Aug/31/2005 )
I am trying to clone a ~120bp fragment into a ~6kb vector. After ligation I transform 50ul DH5a cells with 5ul ligation mix.
Problem 1: I got one colony on my vector alone plate-is this an issue? I got 3-8 colonies on my vector + insert plates
Problem 2: I put up the colonies for miniprep in 5ml LB+Amp (100ug/ml final), let them grow overnight but nothing grew! Incubator is set @37c with shaking.
When you picked the colonies, did you streak on fresh amp plates and did they grow there also?
The freshness of your LB Amp plates might be at fault. I.E. Not enough antibiotic to ward off random bacteria growth. Especially since when you grew them up they did not grow.
When you say your vector alone plate only had one colony, is this cut vector? Or just intact transformed vector?
i'm having the same problem as umm sarah ( i'm just guessing thats your name).
anyways my insert is also about 120 bps. I ordered the oligos for my insert as 8 seperate oligos (sense and antisense-about 40 nts per oligo). Basicly what i did was ligate two annealed oligos at a time (A+B and C+D) for about an hour with T4 DNA ligase at 16 C. I then mixed them togeter and ligated the two previous rxns with more T4 DNA ligase for another hour at 16 C. After that I added the vector (pSliencer -Ambion) and proceeded to ligate them at 1:10 ratio ( 10 fmol:100 fmol). I've varied the vector ligation from 30 mins to 2 hrs at 16 C and like sarah I only get about 2 or 3 colonies, after tranforming them in DH5A cells, that do not contain my insert. In addition those few colonies are able to grow when I plate them on new amp plates.
So I'm wondering if anyone has any idea of how i can improve tansformation efficenicy. I'm wondering if its becasue I'm ligating 4 seperate oligos. ANyways, it'd be great if I can get any feedback.
okay, so after talking to my PI we decided that one of my problems could be that the amp concentration in my liquid is too high. I decreased it to 50ug/ml and put up the colonies again to grow ON. Also, my cultures may not have been aerating (sp?) properly. We'll see what happens. . .
To answer the questions:
1. When I wrote vector alone I meant I did a ligation reaction with just the digested vector, no insert and that when it was transformed into DH5a cells one colony grew.
2. When I did the initial ligation/transformation the resulting colonies were VERY small so I streaked them out on fresh LB+Amp plates to get more to work with. These streaks grew, but not robustly like I have experienced before. I did make sure to streak individual colonies because the colonies on the original transformation plate were very far apart from each other.
I'm pretty sure the plates aren't contaminated because other people in the lab are using them without any problems.
Thanks for the help!!!!
I usually use phosphorylated-oligos when I need to insert into a vector. Also first mix two oligos, heating up at 95C for 5min and cool down to r.t very slowly. Use it immediately for ligation.
It works well.