No Colonies Cloning Vector Competent Cells

[princezz]

Hi, im using porfes-IT, LOOP 4, LOOP 6 etc vectors and Nhe/Nco inserts, i have done gel extraction and have set up my ligation(used T4 buffer), i did the transformation using competent cells made from XL1-Blue and i had no growth i have tried so many times but still no growth on chloramphenicol plates so i was wondering what could be the reasons for my ligation not working?
please help me and thank you for your time.

[pipette_man]

Hi there,
welcome to the forums

I usually do not ligate in volumes more than 10 ul, and not more than 100 ng of dna per reaction.

What ratio of insert:vector are you using 3:1?

Can you post your protocol so we can analye it?

[princezz]

i didnt actually have a protocol all i did was:
for EACH Porfes:
I added 4ul of vector (
2ul of insert (EITHER Nco or Nhe according to the vector)
1ul of T4*10(BUFFER)
0.5ul of T4
mixed it all left the ligation for 1hour at room temperature.
frozen at -20 celcius and used.
I used 5ul of ligation to 50ul of competent cells for transformation and there was NO GROWTH.
So i gathered my ligation is not working??? therefore i want to know why it has gone wrong why my ligation hasnt worked. Thank you for your help.