Protocol Online logo
Top : Forum Archives: : Molecular Biology

Problem with cloning - can't clone a 3.2kb fragment (Jun/29/2005 )

I have been trying to clone a 3.2kb fragment into a 5.5kb vector and it's refusing to work. I digested the vector with Spe I and dephosphorylate with SAP. The insert was released from another plasmid also using Spe I. I used T4 ligase for the reaction and DH5 for my transformation.

I have tried different amount of insert and vector and nothing grew. I've even tried ligating for 3 days at 16 degree and nothing grew. I know my vector and transformation is no problem because I get plenty of colonies if I don't do the SAP treatment, just nothing with my insert.

Does anyone have any tricks to share? Many thanks

-Muly-

Have you run the cut vector and insert on a gel? This is a good way to check for cutting and for the amount of insert and vector -- better than od 260.

-phage434-

Yes. After digestion I always run out my products and clean up the relevent bands using the Qiagen gel extraction kit. I have also tried running out a small amount after gel extraction to check how much DNA I get back. Then I would use about 3ul of vector and upto 30ul of insert (judging from the gel this is about vector:insert ration of 1:10). I have also try other insert amounts and nothing works...sigh.

-Muly-

Hi Muly,

One possibility is that you're problem lies here:

"Then I would use about 3ul of vector and upto 30ul of insert (judging from the gel this is about vector:insert ration of 1:10). "

I tend to find that large volume ligations don't work efficiently (20uL is my absolute top amount). What I would do is add your desired insert and vector together and then EtOH precipitate and resuspend the pellet in 10uL to do the ligation. I tend to find that a higher concentration rather than absolute amounts promotes ligation.

Hope this helps

Scott

-Scott-

You could also be having trouble with DNA damage from UV during your band picking and cleanup.
Make sure you are using a long wave (365 nm) UV lamp or (less good) a 305 nm lamp for very short times. A 254 nm UV lamp will trash your DNA irrecoverably very quickly.

-phage434-

my transformation's problems were linked to the use of UV and EtBr running the gel after QIAquick extraction.

Whene I decide to avoid UV and EtBr transformations worked

-Dees-