Colony PCR screening without desired result

[pisces]

Hi all. I am an undergraduate student which need to finish my final year project before March. Hope you all can help me regarding the problem i face currently.

My final year project is about cloning which my step now stop at colony PCR screening there. So sad because i keep repeating this step after ligation and transformation step. Sometimes the plate got around 10-20 colonies, sometimes 3-10 colonies. I try screen using colony PCR screening but always get negative result without my desire band.

I already do agarose gel electrophoresis and i am sure that my insert and plasmid are digested with 2 different restriction enzyme.

If you think there is any mistake i may done please reply me as soon as possible. Thanks a lot.

[atin]

Pisces!
What protocol are you following for colony PCR? It is very crucial to ensure complete cell lysis for successful colony PCR reaction. On the other hand, If you are sure that you are getting a recombinant plasmid WITH your desired insert, then you might not need to go for a coarse technique like colony PCR. Just use your plasmid as the template and carry out PCR. If your Plasmid is a recombinant one, You'll definitely get a band!
Best of Luck!!

[pisces]

Atin,

Hi. Thanks. I also doing like what you said, using plasmid as template. But the result is no band. Means that there is no insert in my vector. Although i try many times for ligation and transformation, the colonies i get always contain no insert. After this, I will need to repeat all over again starting from DNA extraction after discuss with my lecturer. If i still meet any problem will post out again. Thanks again.
Have a nice day )

[atin]

Pisces!
Your guide might suggest you to go for Alkaline phosphatase treatment to prevent self ligation.. anyways I wanted to tell you that I am facing a similar kind of problem using a double digested plasmid. However, my vector does not self ligate and I dont get ANY recombinant colnies!!!
Do tell me what your guide suggest! that could help me as well!!

[pisces]

Atin,

Hi~ Thanks. Since until now i did not get my desired insert from my colonies, so i am not sure what the problem really is. Actually the 1st time i clone i manage to get the insert i want from one of four colonies grow on LB agar plate (with amp) but after send for sequencing there is some sequence missing and thus i need to repeat my project. After 3 months, i get no result...
U got check your restriction enzyme? I think it might be a problem because i used to change my RE brand before and after that i never get colony with correct insert...
For next trial, i will change to my 1st time use RE, and maybe will prepare new competent cell as suggested by my lecturer. I am quite sure that my vector did not ligate itself because i got do negative control for ligation (which i only put vector, T4 DNA ligase n ligation buffer) and transformation and the result is no colony grow at all.
Hmmm.... seriously, i will never give up if i am given more time but now i only have time until end of march, so i have no choice to give up my cloning if i still fail to get my gene of interest from my colony... My senior suggest me to stop this cloning because he think that the failure i facing now maybe due to my insert gene is too long (2161 bp) but since i had success for the 1st time i think it is not the problem...
Another thing i had tried before is i add ATP in ligation mixture as i got the idea from one of forum but still, it did not work for me...
'Cloning really need luck' is what i heard from my friends.. share it with you..haha.. so, GOOD LUCK to you! I believe that it will work if the primer is ok and all the reagent work well. ALL the best