Transformation, no colonies! - (Jul/10/2008 )

Hi everyone,

I've been reading some of the helpful hints on this site for a few days now and thought I would ask for some help. I have recently moved to a new lab and am having trouble with cloning, which I didnt seem to have in my old lab. (I see I'm not the only one with this problem....)

My problem does not appear to be the ligation because positive control does not grow when transformed either! Here is what I do:

Based on intensity of gel purified product and size I figured the ratios for my vector to insert:

4ul Vector
2ul Insert
1ul Ligase buffer (invitrogen)
1ul T4 DNA Ligase (invitrogen)

I then tranform the entire ligation after incubating at a number of different conditions (overnight at 4degrees, overnight at room temp, 4 hours room temp)

I dont think the ligation is the problem, but I have a suspicion that possibly adding the entire ligation reaction to the competent cells might be killing them? It seems some people will only add 2ul?

I use 100ul competent cells (CaCl2 competent) and add 8ul ligation
-in ice for 30 mins
-heat shock at 42 degrees for 90 seconds
-directly back in ice for 2 mins
-add 500ul LB (no antibiotic)
-shake at 37degrees for 1 hour
-spin at 5,000 for 2 minutes
-remove all but 100ul LB and resuspend pellet in remaining (i've also tried by removing all of LB and resuspending in 100ul fresh LB)
-plate 100ul on prewarmed LB+AMP plates
-incubate overnight and watch as no colonies grow and I proceed to rip my hair out in the morning!!

Any suggestions? Is it possible my ligation is lethal? it seems that i get growth after shaking for 1 hour at 37 degrees but its hard to see if it is "more cloudy"

Anything would be very helpful!!!

Hello!!!

I dont think the relation ligation/ul de cells is incorrect. If you are not the only one that have problem with this, you can think in some of the solutions and the most important: the cells.
Are you sure they are competent?, put them to grow in lb without atb.
good luck!!

so well, what are the molar relation that you are using?!...

50-100 ng vector * kb insert * relation (3/1, 1/1, 1/3, etc)/ kb of vector.= ng of insert

Be carefull, when the insert is so big you should use more insert that vector and viceversa.
The protocol do not seems to be wrong.
Are you sure that the ligase that you are using is ok??..

Try to transform only ciruclar vector and see the results!

good luck

AH!! i forgot!!

the volumen of the ligation must be as 10% of the volumen of the cells. you are using 8ul for 100ul. Itis ok , i have used 20ul for 50ul of cells and it works
perfectly.. so...

Hi, One of the suggestions you see earlier is correct, the insert: vector is about 3:1. You always need more insert than the vector.

In transformation, even 1:25 works. So 2 ul of your ligation mix in 50ul of cells gives good colonies. You can actually do a test for this: use 4 different volumes of the ligation mix: 1, 2, 4, and 8 uls. Transform them in 50ul of cells at the same time. It will be a good test to check what is a better ligation mix vol to transform.

HI THERE! I had my own cloning blues for months until today,, when I got my correct colonies.. I had tried a lot of different stuff but what worked for me I guess, is keeping the ligation reaction(1ul ligase,1ul lig buffer, and the ETOH precipitate of insert and vector mixed together)...for 2 days at 4 C and then transform...might as well try that if nothing else works... Good luck!