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

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.

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.

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

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.

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